Methods for using mutant RNA polymerases with reduced discrimination between non-canonical and canonical nucleoside triphosphates

ABSTRACT

A method for synthesizing a nucleic acid molecule comprising at least one non-canonical nucleoside triphosphate using a mutant polymerase having a reduced discrimination between canonical and non-canonical substrates is disclosed. The method comprises incubating a template nucleic acid in a reaction mixture comprising the mutant nucleic acid polymerase and the appropriate canonical and non-canonical nucleoside triphosphates which are desired substrates for the mutant nucleic acid polymerase. The present invention is also a method of determining the sequence of a nucleic acid molecule using the mutant polymerase to create a nucleic acid molecule comprising at least one non-canonical nucleoside triphosphate.

FIELD OF INVENTION

[0001] The field of the present invention is methods for producingnucleic acid molecules containing at least one non-canonical nucleotideand for characterizing nucleic acid molecules by synthesizing nucleicacid molecules containing at least one non-canonical nucleotide in vitrousing mutant nucleic acid polymerases having at least a 10-fold reduceddiscrimination between 2′-deoxyribonucleoside-5′-triphosphates andribonucleoside-5′-triphosphates as substrates compared to thecorresponding wild-type enzymes.

BACKGROUND

[0002] There are a number of procedures commonly used in the art for invitro synthesis of nucleic acid molecules, including both DNA and RNA.For example, one may use an in vitro transcription reaction tosynthesize RNA from a DNA template present in the reaction. T7-type RNApolymerases, such as T7 RNA polymerase, T3 RNA polymerase or SP6 RNApolymerase, are commonly used in such reactions, although many other RNApolymerases may also be used. Usually, but not always, synthesis of RNAis de novo (i.e., unprimed), and usually, but not always, transcriptionis initiated at a sequence in the template that is specificallyrecognized by the RNA polymerase, termed a “promoter” or a “promotersequence”. A method for in vitro transcription is presented herein.

[0003] Procedures for in vitro nucleic acid synthesis are also commonlyused in the art to amplify nucleic acid molecules, including both DNAand RNA. For example, transcriptions using RNA polymerases are anintegral part of “nucleic acid sequence-based amplification” (NASBA),“self-sustained sequence replication” (3SR), and “transcription-mediatedamplification” (TMA) Hill, C. S., 1996, three similar methods foramplifying nucleic acid molecules in vitro.

[0004] By way of example, all or a specific portion of an RNA moleculemay be amplified using NASBA (Compton, et al., 1991) or 3SR (Fahy, etal., 1991) by isothermal incubation of a sample RNA in a buffercontaining two primers (a first primer complementary to the RNA moleculeand encoding a promoter sequence for an RNA polymerase and a secondprimer complementary to the 3′-end of the first cDNA strand resultingfrom reverse transcription of the RNA molecule), an RNA- andDNA-dependent DNA polymerase which also has RNase H activity (or aseparate RNase H enzyme), all four canonical2′-deoxynucleoside-5′-triphosphates (dATP, dCTP, dGTP and dTTP), an RNApolymerase that recognizes the promoter sequence of the first primer,and all four canonical ribonucleoside-5′-triphosphates (rATP, rCTP, rGTPand rUTP).

[0005] A first cDNA strand is synthesized by extension of the firstprimer by reverse transcription. Then, the RNase H digests the RNA ofthe resulting DNA:RNA hybrid, and the second primer primes synthesis ofthe second cDNA strand. The RNA polymerase then transcribes theresultant double-stranded DNA (ds-DNA) molecule from the RNA polymerasepromoter sequence, making many more copies of RNA, which in turn, arereversed transcribed into cDNA and the process begins all over again.This series of reactions, from ds-DNA through RNA intermediates to moreds-DNA, continues in a self-sustained way until reaction components areexhausted or the enzymes are inactivated. DNA samples can also beamplified by other variations of NASBA or 3SR or TMA.

[0006] Another nucleic acid amplification method involving DNA synthesisis the polymerase chain reaction (PCR).

[0007] By way of example, a specific portion of a DNA molecule may beamplified using PCR by temperature cycling of a sample DNA in a buffercontaining two primers (one primer complementary to each of the DNAstrands and which, together, flank the DNA sequence of interest), athermostable DNA polymerase, and all four canonical2′-deoxynucleoside-5′-triphosphates (dATP, dCTP, dGTP and dTTP). Thespecific nucleic acid sequence is geometrically amplified during each ofabout 30 cycles of denaturation (e.g., at 95° C.), annealing of the twoprimers (e.g., at 55° C. ), and extension of the primers by the DNApolymerase (e.g., at 70° C.), so that up to about a billion copies ofthe nucleic acid sequence are obtained. RNA may be similarly amplifiedusing one of several protocols for (reverse transcription-PCR) RT-PCR,such as, for example, by carrying out the reaction using a thermostableDNA polymerase which also has reverse transcriptase activity (Myers andGelfand, 1991).

[0008] The polymerase chain reaction, discussed above, is the subject ofnumerous publications and patents, including, for example: Mullis, K.B., et al., U. S. Pat. No. 4,683,202 and U. S. Pat. No. 4,965,188.

[0009] A variety of procedures for using in vitro nucleic acid synthesisfor characterizing nucleic acid molecules, including both DNA and RNA,also are known in the art.

[0010] There are many reasons for characterizing nucleic acid molecules.For example, genes are rapidly being identified and characterized whichare causative or related to many human, animal and plant diseases. Evenwithin any particular gene, numerous mutations are being identified thatare responsible for particular pathological conditions. Thus, althoughmany methods for detection of both known and unknown mutations have beendeveloped (e.g., see Cotton, 1993), our growing knowledge of human andother genomes makes it increasingly important to develop new, better,and faster methods for characterizing nucleic acids. Besides diagnosticuses, improved methods for rapidly characterizing nucleic acids willalso be useful in many other areas, including human forensics, paternitytesting, animal and plant breeding, tissue typing, screening forsmuggling of endangered species, and biological research.

[0011] One of the most informative ways to characterize a DNA moleculeis to determine its nucleotide sequence. The most commonly used methodfor sequencing DNA at this time (Sanger, et al., 1977) uses a DNApolymerase to produce differently sized fragments depending on thepositions (sequence) of the four bases (A=Adenine; C=Cytidine;G=Guanine; and T=Thymine) within the DNA to be sequenced. In thismethod, the DNA to be sequenced is used as a template for in vitro DNAsynthesis. RNA may also be used as a template if a polymerase withRNA-directed DNA polymerase (i. e., reverse transcriptase) activity isused. In addition to all four of the deoxynucleotides (DATP, dCTP, dGTPand dTTP), a 2′,3′-dideoxynucleotide is also included in each in vitroDNA synthesis reaction at a concentration that will result in randomsubstitution of a small percentage of a normal nucleotide by thecorresponding dideoxynucleotide. Thus, each DNA synthesis reactionyields a mixture of DNA fragments of different lengths corresponding tochain termination wherever the dideoxynucleotide was incorporated inplace of the normal deoxynucleotide.

[0012] The DNA fragments are labelled, either radioactively ornon-radioactively, by one of several methods known in the art and thelabel(s) may be incorporated into the DNA by extension of a labelledprimer, or by incorporation of a labelled deoxy- or dideoxy- nucleotide.By carrying out DNA synthesis reactions for each of the fourdideoxynucleotides (ddATP, ddCTP, ddGTP or ddTTP), then separating theproducts of each reaction in adjacent lanes of a denaturingpolyacrylamide gel or in the same lane of a gel if differentdistinguishable labels are used for each reaction, and detecting thoseproducts by one of several methods, the sequence of the DNA template canbe read directly. Radioactively-labelled products may be read byanalyzing an exposed X-ray film. Alternatively, other methods commonlyknown in the art for detecting DNA molecules labelled with fluorescent,chemiluminescent or other non-radioactive moieties may be used.

[0013] Because 2′,3′-dideoxynucleotides (ddNTPs), including even ddNTPswith modified nucleic acid bases, can be used as substrates for many DNApolymerases, Sanger's dideoxy-sequencing method is widely used.Recently, Tabor and Richardson (EP application 942034331, 1994) reportedthat mutations at specific sites in many DNA polymerases improved theability of these mutant enzymes to accept ddNTPs as substrates, therebyleading to improved DNA polymerases for DNA sequencing using the Sangermethod.

[0014] Nucleic acid sequencing provides the highest degree of certaintyas to the identity of a particular nucleic acid. Also, nucleic acidsequencing permits one to detect mutations in a gene even if the site ofthe mutation is unknown. Sequencing data may even provide enoughinformation to permit an estimation of the clinical significance of aparticular mutation or of a variation in the sequence.

[0015] Cycle sequencing is a variation of Sanger sequencing thatachieves a linear amplification of the sequencing signal by using athermostable DNA polymerase and repeating chain terminating DNAsynthesis during each of multiple rounds of denaturation of a templateDNA (e.g., at 95° C.), annealing of a single primer oligonucleotide(e.g., at 55° C.), and extension of the primer (e.g., at 70° C.).

[0016] Other methods for sequencing nucleic acids are also known besidesthe Sanger method. For example, Barnes described a method for sequencingDNA by partial ribo-substitution (Barnes, W. M., 1977). In this method,a pre-labelled primer was extended in vitro on a template DNA to besequenced in each of four reactions containing a wild-type DNApolymerase in the presence of Mn2+, all four canonical2′-deoxyribonucleoside triphosphates, and one of four ribonucleosidetriphosphates under deoxy-/ribo-nucleotide ratios and conditions thatresult in about 2% ribonucleotide substitution at each position. Afteralkali treatment to cleave the synthetic DNA at the positions of partialribosubstitution, the sequence was determined by analyzing the fragmentsresulting from each reaction following electrophoresis on a denaturingpolyacrylamide gel.

[0017] Although most methods for sequencing nucleic acids employ DNApolymerases, some work has also been reported on the use of T7 RNAP andSP6 RNAP for transcription sequencing of DNA templates beginning at therespective T7 or SP6 promoter sequence using3′-deoxyribonucleoside-5′-triphosphates (Axelrod, V. D., and Kramer, F.R., 1985), and Q-Beta replicase for sequencing single-stranded RNAtemplates (Kramer, F. R., and Mills, D. R., 1978). Also,3′-O-methyl-ribonucleoside-5′-triphosphates have been used forsequencing DNA templates with E. coli RNA polymerase ((Axelrod, V. D.,et al., 1978). None of these techniques is commonly used at present,perhaps in part, due to the difficulty to obtain the 3′-deoxy- and3′-O-methyl-nucleoside triphosphate substrates, while2′,3′-dideoxy-ribonucleoside-5′-triphosphates that are commerciallyavailable have not been found to be substrates for wild-type (w.t.) RNApolymerases.

[0018] In view of the numerous applications involving in vitro nucleicacid synthesis known in the art, it is useful to consider the propertiesof the key nucleic acid polymerase reagents which make these procedurespossible, and which, if modified in their essential properties, mightimprove these procedures.

[0019] One classification of nucleic acid polymerases relies on theirdifferent template specificities (RNA or DNA), substrate specificities(rNTPs or dNTPs), and mode of initiation (de novo or primed). Thesedesignations usually refer to the template and substrate specificitiesdisplayed in vivo during the fulfillment of a polymerase's biologicalfunction.

[0020] In vitro, polymerases can display novel activities, albeit withreduced efficiency and/or under non-physiological conditions. E. coliDNA-directed DNA polymerase I, for example, can use RNA as a template,although there is a concomitant ˜100-fold increase in dNTP K_(m)(Ricchetti and Buc, 1993). T7 DNA-directed RNA polymerase can also useRNA as a template (Konarska and Sharp, 1989). These are not exceptionalobservations because it is a general property of polymerases that theydisplay relaxed template specificity, at least in vitro.

[0021] While template specificity may be relaxed, polymerase substratespecificity is normally extremely stringent. T7 DNAP, for example,displays at least 2,000-fold selectivity for dNTPs over rNTPs, even inMn++ buffer which relaxes the ability of the polymerase to discriminatebetween dNTPs and ddNTPs (Tabor and Richardson, 1989).

[0022] It has been reported that transcripts synthesized by a T7 RNAPY639F mutant in vivo yielded ½-⅓ of the protein per transcript comparedto transcripts synthesized by the wild-type enzyme (Makarova, et al.,1995). The latter phenotype was unique to the Y639F mutant amongst anumber of other active site mutants examined for in vivo expression, andindicated that Y639F transcripts contained a defect that led to theirbeing inefficiently translated.

[0023] A polymerase with an altered substrate specificity would beuseful in many molecular biological applications, such as creating anucleic acid molecule comprising at least one non-canonical nucleotide.

SUMMARY OF THE INVENTION

[0024] We disclose herein the identification of mutant polymerases, suchas T7-type RNAPs, that display the ability to use dNTPs. The mutationsoccur in tyrosine 639 within motif B (Delarue, et al., 1990) of T7 RNAP.

[0025] We have characterized the ability of the Y639 mutants, as well asa large number of other active site mutants, to use dNTPs in both Mg++and Mn++ buffers. Our results point to a specialized role for tyrosine639 in T7 RNAP—and the corresponding amino acid in other polymerases—ininsuring that substrates to be added to the growing nucleic acid havethe correct structure. The results reveal that both transcript andsubstrate structure affect the efficiency with which the transcript isextended and show that the restriction of unprimed initiation to RNApolymerases is not due to an intrinsic property of ribo- vs.deoxynucleotides, but simply to the selectivity of the polymerase activesite. The present invention provides researchers with novel polymerasereagents and improved methods that expand the structural range ofnucleic acids that can be enzymatically synthesized in vitro.

[0026] The present invention requires a polymerase with a reduceddiscrimination between canonical and non-canonical nucleosidetriphosphates. In a preferred embodiment of the present invention, thepolymerase has a reduced discrimination between rNTPs and dNTPs. In anespecially preferred embodiment, the reduced discrimination is at least10-fold compared to wild-type enzymes.

[0027] In one embodiment, the present invention is a method forsynthesizing a nucleic acid molecule that comprises at least onenon-canonical nucleotide. This method comprises the steps of incubatinga template nucleic acid in a reaction mixture suitable for nucleic acidpolymerization containing a mutant nucleic acid polymerase and theappropriate canonical and non-canonical nucleoside triphosphates whichare substrates for a mutant nucleic acid polymerase and which aredesired to be incorporated into the synthesized nucleic acid molecule.

[0028] In an especially preferred form of this method, the synthesizednucleic acid molecule has an altered susceptibility to a nucleasecompared to a nucleic acid which could be synthesized using thecorresponding non-mutant nucleic acid polymerase with canonicalnucleoside triphosphates.

[0029] The present invention is also a method for determining thesequence of a nucleic acid molecule using a mutant RNA polymerase.

[0030] The method comprises synthesizing a nucleic acid molecule, eitherde novo from a promoter, or by extending a primer annealed to thetemplate molecule in four separate reactions. The four separatereactions each have all 4 rNTPs and a portion of a ddNTP, or have all 4dNTPs and a portion of a ddNTP, or have 4 2′-fluorine-substituted NTPsand a portion of a ddNTP. Chain termination will occur and the productsmay be evaluated so that the sequence of the template molecule may bededuced. In one embodiment of this method, the reactions which include addNTP occur in the same reaction mixture and are linked to a method fornucleic acid amplification, including, but not limited to, NASBA, 3SR,TMA, or other similar methods.

[0031] The present invention is also a partial ribo-substitution methodfor determining the sequence of a nucleic acid molecule. This methodcomprises synthesizing a nucleic acid molecule, either de novo from apromoter or by extending a primer annealed to the template molecule infour separate reactions. The reactions each have, either four dNTPs anda portion of an rNTP or four 2′-F-NTPs and a portion of an rNTP, or fourdifferent non-canonical nucleoside triphosphates, wherein thesenucleoside triphosphates have substituents different than a hydroxylgroup at the 2′ position of the ribose and which the mutant polymerasecan use as substrates for synthesis in nucleic acids, and a portion ofan rNTP. The reaction products are then cleaved at sites containing anincorporated rNTP by using an alkaline solution or an RNase, and thecleaved nucleic acid fragments are separated according to size so thatthe sequence of the template molecule may be determined.

[0032] The present invention is also embodiments of a partialribo-substitution method wherein the nucleic acid synthesis reactions ofsaid method occur in the same reaction mixture and are also part of orlinked to a method for nucleic acid amplification, including, but notlimited to, NASBA, 3SR, TMA, or other similar methods.

[0033] In still other embodiments of the present invention, the productsof either 1, 2, 3, or 4 of the dideoxy-sequencing reactions or of thepartial ribo-substitution sequencing reactions are performed or analyzedto determine the presence or absence of a particular nucleic acid, orits relatedness to another nucleic acid, or whether it contains amutation compared to another nucleic acid.

[0034] The present invention is also a kit for performing any of theabove-identified methods.

[0035] It is an object of the present invention to provide a mutantpolymerase capable of altered discrimination between canonical andnon-canonical nucleoside triphosphates.

[0036] It is an object of the present invention to provide an improvedDNA sequencing method.

[0037] It is an object of the present invention to provide a method todetect the presence of a nucleic acid.

[0038] It is an object of the present invention to provide a method todetect the identity of a nucleic acid.

[0039] It is an object of the present invention to provide a method todetect mutations in a nucleic acid.

[0040] It is an object of the present invention to minimize the stepsinvolved in amplifying and sequencing, detecting, identifying anddetecting mutations in nucleic acids.

[0041] It is another object of the present invention to provide a methodfor synthesizing nucleic acid molecules with altered nucleasesusceptibility.

[0042] It is another object of the present invention to provide a methodfor synthesizing nucleic acid molecules comprising at least onenon-canonical nucleoside triphosphate.

[0043] Other objects, features and advantages of the present inventionwill become apparent after examination of the specification, claims anddrawings.

DESCRIPTION OF THE DRAWINGS

[0044]FIG. 1 diagrams the transcription products produced by Y639F andw.t. T7 RNAP in the presence of various combinations of rNTPs and dNTPs.

[0045]FIG. 2 shows the effect of dGTP substitution on transcription bythe w.t. and Y639F polymerase.

[0046]FIG. 3 shows the effects of single-strandedness and sequence inthe initially transcribed region on the activity of Y639F in reactionswith 4 rNTPs or 4 dNTPs.

[0047]FIG. 4 shows transcription by Y639F and w.t. polymerase with dGTPor rGTP on poly(dI)·poly (dC).

[0048]FIG. 5 shows primed synthesis of DNA and RNA with Y639F and thew.t. polymerase.

[0049]FIG. 6 shows relative elongation rates of Y639F in “4 rNTP” and “3rNTP+1 dNTP” reactions.

[0050]FIG. 7 is a diagram of the mutagenesis strategy involved increating a mutant SP6 RNA polymerase.

DESCRIPTION OF THE INVENTION Definitions

[0051] By “mutant polymerase” is meant a nucleic acid polymerase whichhas at least one altered amino acid compared to the correspondingwild-type polymerase, wherein said mutation or alteration results in themutant polymerase having a reduced discrimination between non-canonicaland canonical nucleoside triphosphates as substrates.

[0052] By “template” we mean a macromolecular pattern or mold for thesynthesis of another macromolecule, composed of a sequence ofnucleotides, either rNTPs or dNTPs, that serves to specify thenucleotide sequence of another structure. “Nucleotide” refers to abase-sugar-phosphate compound. Nucleotides are the monomeric subunits ofboth types of nucleic acid polymers, RNA and DNA. “Nucleotide” refers toribonucleoside triphosphates, rATP, rGTP, rUTP and rCTP, anddeoxyribonucleoside triphosphates, such as DATP, dGTP, dTTP, dCTP.

[0053] As used herein, “nucleoside” refers to a base-sugar combinationwithout a phosphate group. “Base” refers to the nitrogen-containingbase, for example adenine (A), cytidine (C), guanine (G) and thymine (T)and uracil (U). “Incorporation” refers to becoming a part of a nucleicacid polymer. There is a known flexibility in the terminology aboutincorporation of nucleic acid precursors. For example, the nucleotidedGTP is a deoxyribonucleoside triphosphate. Upon incorporation into DNA,it becomes a dGMP, or deoxyguanosine monophosphate moiety. Althoughthere is no dGTP molecule in DNA, one may say that one incorporates dGTPinto DNA.

[0054] As defined herein, a “canonical” nucleoside triphosphate for anRNA polymerase (“RNAP”) consists of any ribonucleoside-5′-triphosphate(“rNTP” or “NTP”) which has an hydroxyl group at the 2′-position of thesugar, including, but not limited to, the four common ribose-containingsubstrates for an RNA polymerase -ATP, CTP, GTP and UTP. A2′-deoxyribonucleoside-5′-triphosphate (“dNTP”) which has hydrogen atthe 2′-position of the sugar, including, but not limited to, the fourcommon deoxyribose-containing substrates (dATP, dCTP, dGTP and dTTP,also known as “TTP”) for a DNA polymerase (“DNAP”) is defined herein asa “non-canonical” nucleoside-5′-triphosphate or a “non-canonical NTP” ora “non-canonical nucleotide” or a “non-canonical deoxynucleotide” or a“non-canonical triphosphate” or a “non-canonical substrate” for an RNApolymerase. On the other hand, a “canonical” nucleoside triphosphate fora DNAP consists of any dNTP which has a hydrogen at the 2′-position ofthe sugar, while an rNTP is defined as a “non-canonical NTP” or a“non-canonical nucleotide” or a “non-canonical substrate” for a DNAP.The terms “canonical” and ′non-canonical“are meant to be used hereinonly with reference to the 2′ position of the sugar. Thus, as definedherein, 2′,3′-dideoxynucleoside-5′-triphosphates (” 2′,3′-ddNTPs” or“ddNTPs”) are “non-canonical” substrates for an RNAP, but are defined as“canonical” for a DNAP. Further, any other substituent than an hydroxylgroup at the 2′-position of ribose or a hydrogen at the 2′-position ofdeoxyribose, including, but not limited to, a fluorine (“F” or “fluoro”group) or an amino group, would be defined as “non-canonical” for bothRNAPs and DNAPs herein. The terms “canonical” or “non-canonical” alsoare not meant to refer to the nucleic acid bases attached to the sugarmoieties. Thus, for example, other natural or modified nucleic acidbases attached to the 1′-position of ribose-5′-triphosphate would stillbe defined as “canonical” herein.

[0055] By “a (mutant) nucleic acid polymerase (enzyme) with reduceddiscrimination between canonical and non-canonical nucleosidetriphosphate substrates”, we have a specific quantitative definitioncalculated as follows:

[0056] 1. One first determines the K_(m) and the k_(cat) for each enzyme(mutant and wild-type) using the non-canonical nucleotide as a substrateand using the canonical nucleotide as a substrate, as was describedpreviously (Patra, et al., 1992). The value “K_(m) ” expresses howreadily the enzyme will bind the substrate (a larger K_(m) impliesweaker binding) and “k_(cat)” expresses the rapidity with which thesubstrate, once bound by the enzyme, is reacted upon.

[0057] 2. One next calculates the numerical value for k_(cat)/K_(m) foreach enzyme and for each substrate. By broad scientific consensus, thespecificity of an enzyme for a substrate is felt to be most suitablyexpressed by this ratio.

[0058] 3. For each enzyme, one then calculates the numerical valueobtained by using the value of k_(cat)/K_(m) for the canonical substratein the numerator and k_(cat)/K_(m) for the non-canonical substrate inthe denominator. This number indicates how much a given enzymediscriminates between the two substrates. For example, if this valueequals 1, then the enzyme uses both the canonical and the non-canonicalsubstrates equally well; it does not discriminate between the twosubstrates. If this value is greater than 1, then the enzymediscriminates by that factor between the two substrates; for example, ifthe value is 100, then the enzyme discriminates by a factor of 100 infavor of the canonical substrate compared to the non-canonicalsubstrate. Similarly, a value less than 1 means that the enzymediscriminates in favor of the non-canonical substrate over the canonicalsubstrate.

[0059] 4. Finally, one compares the numerical value calculated in step 3above for the wild-type (w.t.) enzyme with the value calculated in step3 for the mutant enzyme. If the value calculated for the mutant enzymeis less than the value calculated for the wild-type enzyme, then themutant enzyme “has a reduced discrimination for the non-canonicalsubstrate compared to the wild-type enzyme. ” For example, if the valuescalculated in step 3 are 100 for the wild-type enzyme and 10 for themutant enzyme, then the discrimination of the mutant enzyme in favor ofthe canonical substrate (or against the non-canonical substrate) isreduced 10-fold compared to the discrimination of the wild-type enzyme.

[0060] We have found that for wild-type T7 RNAP, the average of thek_(cat)/K_(m) values for the four common rNTPs (ATP, CTP, GTP & UTP) isabout 120-fold larger than the average k_(cat)/K_(m) values for the fourcommon dNTPs (dATP, dCTP, dGTP and dTTP); i.e., the wild-type enzymediscriminates by a factor of 120 for rNTPs vs. dNTPs. For the Y639Fmutant enzyme, the average of the k_(cat)/K_(m) values for the fourcommon rNTPs is only about 6-fold larger than the average k_(cat)/K_(m)values for the four common dNTPs. Thus, using the average k_(cat)/K_(m)values for these substrates, the Y639F mutant T7 RNAP enzyme has about a20-fold reduced discrimination between dNTPs and rNTPs. However, it isrecognized that the difference in discrimination between wild-type andmutant enzymes will vary depending on the non-canonical substrates andthe mutant enzymes used. Therefore, for the purposes of this invention,we herein define “a (mutant) nucleic acid polymerase (enzyme) withreduced discrimination between canonical and non-canonical nucleosidetriphosphate substrates” as “a polymerase which has at least a 10-foldreduced discrimination compared to the corresponding wild-type enzymefor non-canonical nucleotides compared to canonical nucleotides, whereinthe respective values for discrimination between canonical andnon-canonical substrates is calculated using the average of thek_(cat)/K_(m) values for all four rNTPs and all four dNTPs.”

[0061] By “T7-type RNA polymerases” we mean T7, T3, φI, φIIH, W31, ghl,Y, A1122, SP6 and mitochondrial RNAPs.

In General

[0062] There are many reasons to synthesize nucleic acid moleculescontaining at least one non-canonical nucleotide. For example,incorporation of a non-canonical nucleotide may make the syntheticnucleic acid more resistant and therefore, more stable, to a nuclease,such as a ribonuclease. Also, one may wish to incorporate one or morenon-canonical nucleotides which, for example, change the nucleasedigestion pattern so that the product nucleic acid is easier to detector characterize. For example, because RNase A cleaves RNA only after Cor U, replacement of one or both of these rNMPs by a dNMP or othernon-canonical nucleotide that is resistant to cleavage by RNase A wouldalter the digestion pattern of the nucleic acid.

[0063] There are many uses for nucleic acids which have one or more ofthese properties, such as nuclease resistance. For example, nucleicacids containing at least one non-canonical nucleotide may haveadvantages for use as ribozymes, or as nucleic acid molecules used forgene therapy, in a vaccine, in an antiviral composition, in anantimicrobial composition, in an anti-sense composition for regulatinggene expression, in a composition for hybridization to a complementarynucleic acid, including as a primer, or as a probe for detection of acomplementary nucleic acid for a variety of purposes.

[0064] Some nucleic acid molecules, such as those of mixed dNMP/rNMPcomposition, are highly useful for certain applications, but arepresently difficult or impossible to produce on a practical scale. Thus,improved methods for synthesizing such nucleic acid molecules in vitrowould be highly desirable. For example, probes of mixed DNA-RNA-DNAcomposition for the Cycling Probe Assay (Duck, P. G., et al., 1990) arecurrently made using difficult chemical methods.

[0065] We describe herein previously-unknown properties of T7-type RNApolymerases having a non-wild-type amino acid at specific positionswithin the polypeptides. We found that altering the amino acid at thesespecific positions results in mutant polymerases having at least a10-fold reduced discrimination between2′-deoxyribonucleoside-5′-triphosphates (dNTPs) andribonucleoside-5′-triphosphates (rNTPs) as substrates in in vitronucleic acid synthesis reactions compared to the corresponding wild-typeenzymes. We found that these mutant polymerases also have reduceddiscrimination for other non-canonical nucleoside triphosphate (NTP)substrates, including 2′,3′-dideoxy-ribonucleoside-5′-triphosphates(ddNTPs) and 2′-fluoro-nucleoside-5′-triphosphates (2′-F-NTPs). Based onknowledge of these novel properties, we have disclosed methods for usingthese mutant polymerases for producing nucleic acid molecules containingat least one non-canonical nucleotide and for characterizing nucleicacid molecules by synthesizing nucleic acid molecules containing atleast one non-canonical nucleotide in vitro.

[0066] In one preferred embodiment, the invention uses mutant RNApolymerases that efficiently utilize deoxynucleoside triphosphates assubstrates. In vitro, this mutant will synthesize RNA, DNA, or′transcripts′ of mixed dNMP/rNMP composition from a template moleculedepending on the mix of NTPs or dNTPs present in the synthesis reaction.

Mutant Polymerases of the Present Invention

[0067] In a preferred embodiment, the polymerase mutation isconservative, for example, changing tyrosine 639 (of T7 polymerase)within the active site to phenylalanine, and does not substantiallyaffect promoter specificity or overall activity. Non-conservativemutations of this tyrosine also reduce discrimination between deoxy- andribo-nucleoside triphosphates, but these mutations also typically causelarge activity reductions. Among the most active of the non-conservativemutations, enzymes with methionine or leucine in place of the wild-typetyrosine at the 639 position of T7 RNAP had about half the enzymaticactivity of the wild-type enzyme.

[0068] Of 26 other mutations of other amino acid positions examined inand around the active site of T7 RNAP, none showed marked effects onrNTP/dNTP discrimination.

[0069] T7 RNA polymerase can use RNA templates as well as DNA templatesand is capable of both primer extension and de novo initiation. TheY639F mutant, described below in the Examples, retains the ability touse RNA or DNA templates. Thus, this mutant can display de novoinitiated or primed DNA directed DNA polymerase, reverse transcriptase,RNA directed RNA polymerase, or DNA directed RNA polymerase activitiesdepending on the templates and substrates presented to it in thesynthesis reaction.

[0070] A major theme of research on nucleic acid polymerases over thepast several years has been the discovery of extensive structuralsimilarity between the majority of these enzymes, even those fromfunctionally different classes (Sousa, et al., 1993; Pelletier, et al.,1994; Jacob-Molina, et al., 1993; Kohlstaedt, et al., 1992; Sawaya, etal., 1994; Steitz, et al., 1994). One part of this work has been theidentification of well-conserved residues. Five amino acids have beenidentified as invariant in a large number of DNA-directed RNApolymerases (Delarue, et al., 1990). In T7 RNAP these are D537, K631,Y639, G640A and D812. A specific, conserved function has been revealedfor the two invariant aspartates in coordinating the catalytic Mg++ ion(Sousa, et al., 1993; Pelletier, et al., 1994; Jacob-Molina, et al.,1993; Kohlstaedt, et al., 1993; Sawaya, et al., 1994; Steitz, et al.,1994). Our observations imply a similarly specific and conservedfunction for Y639 as a sensor of inappropriate geometry or structure inthe template-NTP-primer/RNA complex.

[0071] The present invention encompasses methods for synthesis ofnucleic acids containing at least one non-canonical nucleotide usingmutant nucleic acid polymerases which have reduced discrimination fornon-canonical nucleoside triphosphate substrates. The examples belowdemonstrate the reduced dNTP/rNTP discrimination of mutants of T7 RNApolymerase and SP6 RNA polymerase. Genes for other polymerases may bemodified or mutated to obtain mutant enzymes which have similar reduceddiscrimination for non-canonical substrates. If one wished to mutate anRNA

[0072] polymerase to have the properties described herein, one wouldfirst locate the amino acid corresponding to the T7 polymerase Y639 inother RNA polymerases. Identification of the corresponding mutation sitein other polymerases can be done by the well-established procedure ofsequence alignment, which involves aligning the amino acid sequences oftwo proteins, introducing gaps and insertions, and shifting thesequences with respect to each other while maintaining their originallinearity. Such alignment procedures are often performed with the aid ofone or more computer programs into which the amino acid sequences thatone wishes to compare have been entered. When the sequence identity oftwo proteins is high enough (greater than or equal to 30%) over asufficient length of amino acids (greater than or equal to 50), thisprocedure is very reliable in identifying amino acids that occupycorresponding structural and functional positions in the two proteins.Such conditions are met for the T7-type group of RNAPs, which includeT7, T3, φI, φIIH, W31, ghl, Y, A1122, SP6 and mitochondrial RNAPs, andallow identification of the mutation site corresponding to Y639 in T7RNAP.

[0073] Using this method, we predicted that the amino acid in w.t. SP6RNAP that corresponded to the Y639 site in T7 RNAP was Y631, and asdescribed herein, mutagenesis of this site resulted in a Y631F mutantSP6 RNAP which has a similar reduced discrimination for dNTPs comparedto rNTPs like the Y639F mutant T7 RNAP.

[0074] From alignment studies, it is known that there is a conservedmotif present in T7-like RNAPs and class I DNAPs with the followingconsensus sequences:

[0075] . . . K———————Y G . . .

[0076] where Y is the tyrosine at amino acid number 639 in the T7 RNApolymerase protein. The same consensus sequence is observed in the SP6RNA polymerase and T3 RNA polymerase proteins where a K (K=lysine) issucceeded by 7 amino acids and a Y G (G=glycine). In SP6 RNA polymerasethe Y is at amino acid number 631 in the polypeptide chain, and in T3RNA polymerase it is at amino acid number 573. By mutating the codon forY631 in SP6 RNA polymerase such that a phenylalanine is at thisposition, the expected phenotypic change was realized.

[0077] In summary, one may locate the corresponding mutation site inother RNAPs by aligning the amino acid sequence of a T7-like RNAP,chosen from among T7, T3, φI, φIIH, W31, ghl, Y, A1122, SP6 andmitochondrial RNAPs, against the conserved motif given above andidentifying which position corresponds to the Y639 position in T7 RNAP.

[0078] As stated above, the conserved motif is also present in class IDNAPs. While a structure of T7 RNAP complexed with NTP is not available,the structure of the homologous Klenow fragment of DNAP I with dNTP hasbeen obtained (Beese, et al., 1993). This structure demonstrated thatthe amino acids encompassed within the above-mentioned conserved motif(i.e., amino acid residues 758 to 767 of E. coli DNAP I) are inproximity to the deoxyribose sugar of the dNTP, so that it is reasonablethat mutations within this motif might affect the ability of a DNAP todiscriminate between dNTPs and rNTPs. However, one of the presentinventors found that when a mutation was made which changed the aminoacid at the position in a class I DNAP corresponding to the Y639mutation in T7 RNAP, the mutant DNAP retained enzymatic activity, butdid not have a reduced discrimination for rNTPs compared to dNTPs. Thus,it is not obvious what mutations, if any, would result in a class I DNAPhaving reduced discrimination for rNTPs vs. dNTPs, even if it isreasonable to assume that such a mutation would occur within theabove-mentioned conserved motif (residues 758 to 767 of E. coli DNAP I)which the structure shows to be in proximity to the dNTP.

[0079] Because the structure of the Klenow fragment of DNAP I complexedwith dNTP was determined (Beese, et al., 1993), researchers havebelieved that the homologous conserved motif of T7 RNAP (i.e., aminoacid residues 631-640) is likely to be in proximity to the ribose moietyof an NTP, as the case for the DNAP I. Nevertheless, prior to the workof the present invention, it was not possible to know which mutation, ifany, might result in a reduced discrimination for dNTPs vs. rNTPs. Sincethe Y639 mutation of T7 RNAP was identified, as presented herein, one ofthe present inventors has modeled NTP in T7 RNAP (Huang, et al.,submitted for publication) based on the structures of the homologousKlenow fragment of DNAP I complexed with dNTP (Beese, et al., 1993) andof RT complexed with primer-template (Jacabo-Molina, et al., 1993).Models based on either structure agree in placing the ribose close toY639, and in revealing no other side chain capable of discriminating thehydrogen bonding character of the 2′-substituent within 5 angstroms ofthe 2′-group of the NTP. Thus, the model is consistent with our resultsrelated to a reduced discrimination of Y639 RNAP mutants for dNTPs vs.rNTPs, even though additional studies (Huang, et al., submitted forpublication) have determined that the hydrogen bonding character is notthe only factor involved in dNTP/rNTP discrimination.

[0080] Less is known about non-T7-type RNAPs. For many, the amino acidsequences are not known. Non-phage-encoded host bacterial RNApolymerases are complex multi-subunit proteins. A nucleotidepolymerization site has been localized in the β subunit of E. coli RNApolymerase although participation of other subunits is not ruled out. Inorder to determine the site in a non-T7-like RNAP which would result ina reduced dNTP/rNTP discrimination, one would first use theabove-described procedure of alignment to determine if the . . .K———————Y G . . . motif was present. If so, it may be possible to obtainthe desired mutation in the same manner as for T7-like RNAPs. However,if the conserved motif is not present, one may obtain the desiredmutation with greater difficulty by random mutagenesis and enzyme assayscreening in order to find a change or changes that result in reduceddNTP/rNTP discrimination.

[0081] Once one has determined where the corresponding Y639 site is inthe polymerase one wishes to mutate, one would use standard methods inthe art of molecular biology to create an amino acid substitution. Asdisclosed above, a conservative substitution is preferable. For example,a substitution of a phenylalanine for a tyrosine is most preferable. TheExamples below disclose a method for creating a mutant polymerase, butone of skill in the art will realize that there are many substitutemethods of equal effectiveness.

Methods of the Present Invention

[0082] In one embodiment, the present invention is a method for using amutant polymerase for synthesizing in vitro a nucleic acid moleculewhich comprises at least one non-canonical nucleotide in place of atleast a portion of the canonical nucleotides. The method comprises thesteps of incubating a template nucleic acid in a reaction mixturecontaining a mutant nucleic acid polymerase which has reduceddiscrimination between canonical and non-canonical nucleosidetriphosphates, including between dNTPs and rNTPs, and the appropriatecanonical or non-canonical nucleoside triphosphates which are substratesfor the nucleic acid polymerase. One then follows standard polymerasereaction protocols and creates the synthesized nucleic acid molecule.

[0083] Preferably, the reactions also contain inorganic pyrophosphatase,which is known to increase the yields in in vitro transcriptionreactions (Cunningham, P. R. and Ofengand, J., 1990) and to reducepyrophosphorolysis in in vitro DNA synthesis reactions (Tabor, S., andRichardson, C. C., 1990), as well as buffers and other components whichare known to those of skill in the art to be optimal for the particularw.t. polymerase used. Cunningham and Ofengand (1990) provide an exampleof conditions which may be used for unprimed synthesis with T7 RNApolymerase or mutant T7 RNAPs, although one of skill in the art willrecognize, with respect to reactions with these enzymes or otherenzymes, the need to optimize the concentrations and ratios of canonicaland non-canonical NTP substrates according to the respective K_(m) andapplication and to modify reaction conditions, such as temperature,amount of enzyme, salt concentration, or divalent cation (e.g., Mg2+orMn2+) concentration, in order to produce improved results such as higheryield or a greater percentage of full-length products.

[0084] In a preferred form of this method, the resulting synthesizednucleic acid molecule has a different susceptibility to a nucleasecompared to a nucleic acid synthesized by the corresponding non-mutantnucleic acid polymerase under identical reaction conditions withcanonical substrates. By “different susceptibility” we mean to includereduced, increased, or, in the case of synthetic nucleic acidscontaining both canonical and non-canonical nucleotides, alteredsusceptibility to a nuclease, which may be either a DNAse or RNAse. Thenature of the reduced, increased or altered susceptibility to a nucleaseis also related to the properties of the nuclease. For example, anucleic acid resistant to RNase A, which cleaves RNA only after C or U,may be synthesized using fewer non-canonical nucleotides (e.g., dNTPs or2′-F-NTPs) than a nucleic acid which is resistant to RNase I, whichcleaves after every base.

[0085] In a preferred form of the present invention, the resultingsynthesized nucleic acid is a ribozyme or a nucleic acid molecule usedfor gene therapy, in a vaccine as an antiviral composition, in anantimicrobial composition, as an antisense composition for regulatinggene expression, in a composition for hybridization to a complementarynucleic acid, such as for a primer, or as a probe for detection of acomplementary nucleic acid.

[0086] The resulting synthesized nucleic acid may be either single- ordouble-stranded.

[0087] The present invention is also a kit for performing the method ofsynthesizing a nucleic acid containing at least one non-canonicalnucleotide. Typically, the kit contains a mutant nucleic acid polymerasewith a reduced discrimination for non-canonical compared to canonicalsubstrates and data or information describing conditions under which themethod may be performed.

[0088] The present invention is also improved methods for sequencingnucleic acids using a mutant nucleic acid polymerase of the presentinvention.

[0089] Because 2′,3′-dideoxynucleotides are not substrates for wild-typeRNA polymerase, it previously has not been possible to use the Sangermethod for determining the sequence of a nucleic acid with an RNApolymerase, although 3′-deoxy- or 3′-hydroxymethyl analogs have beenused as terminators for Sanger-like sequencing with RNA polymerases.

[0090] However, 2′,3′-ddNTPs are substrates for the mutant nucleic acidpolymerases of this invention which can also utilize both rNTPs anddNTPs as substrates, and the present invention is also a method forsequencing nucleic acid molecules (DNA or RNA) using a mutant nucleicacid polymerase and 2′,3′-ddNTPs as terminators.

[0091] In one embodiment of this method, the nucleic acid to besequenced, whether DNA or RNA, is used as a template for in vitronucleic acid synthesis from a primer (i.e., primed synthesis) using amutant RNA polymerase which has a reduced discrimination for dNTPscompared to rNTPs. Each of four different reactions also contains anamount of at least one nucleoside triphosphate corresponding to eachnucleic acid base represented in either DNA or RNA, chosen from amongthe 2′-deoxynucleotides DATP, dCTP, dGTP and dTTP or dUTP, or the fourcommon ribonucleotides ATP, CTP, GTP and UTP, or the2′-fluorine-substituted nucleotides 2′-F-ATP, 2′-F-CTP, 2′-F-GTP and2′-F-UTP or 2′-F-TTP. A 2′,3′-dideoxynucleotide is also included in eachin vitro nucleic acid synthesis reaction in an amount that will resultin random substitution by the dideoxynucleotide of a small percentage ofthe corresponding rNTP, dNTP or 2′-F-NTP that is present in the reactionand that would be incorporated into the synthetic nucleic acid in atemplate-dependent fashion.

[0092] Thus, each DNA synthesis reaction yields a mixture of DNAfragments of different lengths corresponding to chain terminationwherever the dideoxynucleotide was incorporated in place of the ribo-,2′-deoxy- or 2′-fluoro- nucleotide. The DNA fragments are labelled,either radioactively or non-radioactively, by one of several methodsknown in the art and the label(s) may be incorporated into the DNA byextension of a labelled primer, or by incorporation of a labelled ribo-,deoxy-, 2′-fluoro- or 2′,3′-dideoxy-nucleotide.

[0093] By carrying out DNA synthesis reactions for each of thedideoxynucleotides (ddATP, ddCTP, ddGTP and ddTTP or ddUTP), thenseparating the products of each reaction in adjacent lanes of adenaturing polyacrylamide gel or in the same lane of the gel ifdifferent distinguishable labels are used for each separate reaction,and then detecting those products by one of several radioactive ornon-radioactive methods known in the art, the sequence of the DNAtemplate can be read directly. Also, other matrices than polyacrylamidewhich separate the fragments based on size may be used. Those with skillin the art will also recognize that other nucleotide analogs generallyused for reducing sequencing compressions, such as ribo-, deoxy- or2′-fluoro- nucleoside triphosphates containing 7-deaza-guanine orinosine, may also be used in place of the ribo-, deoxy- or2′-fluoro-nucleotide for which the respective analog is used.

[0094] The present invention also comprises another embodiment of thismethod for sequencing using 2′,3′-ddNTPs and a mutant RNA polymerasewhich has a reduced discrimination for dNTPs compared to rNTPs. In thisembodiment of the method, the nucleic acid to be sequenced, whether DNAor RNA, is used as a template for de novo (i.e., unprimed) in vitronucleic acid synthesis beginning at an RNA polymerase promoter. In thisembodiment, a primer is omitted from the reactions and depending on thepromoter sequence, in addition to the other components used in the firstembodiment, an amount of a dinucleoside tetraphosphate or atrinucleoside penta-phosphate may be added as an initiating nucleotide(Moroney and Piccirilli, 1991) so that the majority of nucleic acidsynthesis is initiated from a single site. Because no primer is used,the labelling of the sequencing products must be carried out by one ofthe other methods envisioned and discussed with respect to the firstembodiment of the method or by incorporating a label into or on aninitiating dinucleotide or trinucleotide. In other respects, the secondembodiment of this sequencing method of the invention is similar to thefirst embodiment of the method.

[0095] The present invention also comprises methods of sequencingnucleic acids by partial ribo-substitution using mutant nucleic acidpolymerase which have a reduced discrimination between non-canonical andcanonical nucleotides. These methods have advantages over the partialribo-substitution sequencing method described by Barnes (1977), whichrelied on the use of a Mn2+-containing reaction buffer to relax theability of a wild-type DNA polymerase to discriminate between dNTPs andrNTPs. Further, only DNA was used as a template for this method and denovo (i.e., unprimed) nucleic acid synthesis was not envisioned. Incontrast, the ribo-substitution sequencing method of the presentinvention uses a mutant nucleic acid polymerase which has an inherentreduced discrimination between dNTPs and rNTPs and, although it may beincluded, Mn2+ is not required in the sequencing reactions. In stillanother embodiment, 2′-fluorine-substituted NTPs are used in place ofdNTPs in the ribo-substitution reaction. Embodiments of the method ofthe present invention also include sequencing using either DNA or RNA astemplates and sequencing using either nucleic acid primers or de novonucleic acid synthesis from an RNA polymerase promoter sequence.

[0096] Since incorporation of the sequence-delimiting ribonucleotidedoes not terminate nucleic acid synthesis during partialribo-substitution sequencing, all of the radioactive or non-radioactivelabel must be incorporated into the sequencing reaction products priorto incorporation of the first ribonucleotide in order to avoid multiplelabeled produced from each nucleic acid molecule synthesized. Multiplelabeled products, starting from different positions on the template,would make it difficult or impossible to interpret sequence results.Thus, labeling of nucleic acid products for partial ribo-substitutionsequencing must be accomplished by means such as incorporating the labelinto a primer, when used, or into an initiating di- or tri- nucleotide,or by prelabelling in the presence of an amount of a labeled nucleotidewhich will be used up or destroyed and/or limiting 2′-deoxy- or2′-fluoro-nucleotides prior to addition of the sequence-delimitingrNTPs.

[0097] We envision sequencing reactions wherein all of thedistinguishable non-radioactive labels in, or attached to, or connectedwith, the products from more than one of the reactions, up to andincluding all four of the sequencing reactions for a template, or evenreactions from multiple templates if distinguishable non-radioactivelabels are present, are detected in or from a single lane of asequencing gel or a single capillary electrophoresis tube or a singlematrix or means of any kind using an automated sequencer or otherdetection device.

EXAMPLES Example 1: Creation and Characterization of a Mutant T7 RNAPolymerase A. Materials and Methods

[0098] Nucleic acids and NTPs: Nucleotides were from Pharmacia orUSB/Amersham. Polynucleotides were from Pharmacia and the MidlandCertified Reagent Company. Synthetic DNAs were prepared at the UTHSCSADNA synthesis facility on an Applied Biosystems DNA synthesizer andpurified by HPLC. A synthetic RNA 12 mer was from the Midland CertifiedReagent Company. Plasmids pT75 (Tabor and Richardson, 1985) and pBS(Stratagene Inc.) were purified from E. coli by alkaline-lysis andcesium chloride gradient centrifugation (Sambrook, et al., 1989).Radioactive nucleotides were from NEN Dupont or ICN.

[0099] Preparation and purification of mutant polymerases: Construction,expression, and purification of the T7 RNAP mutants was describedpreviously (Bonner, et al., 1992).

[0100] Transcription reactions: Transcription reactions were carried outin 40 mM Tris-Cl pH 8.0, 15 mM MgCl₂, and 5 mM DTT or 20 mM ManganeseCitrate pH 8.0, 5 mM DTT at 37° C. Template, polymerase, and NTPconcentrations were as indicated in the legends to the figures andtables. Relative activity determinations were made by taking 4 μlaliquots of reactions at 5, 10, and 20 minute time points and spottingon to DE81 filter paper. Unincorporated nucleotides were separated fromincorporated nucleotides by washing the filter paper with 0.5 M KH₂PO₄pH 7. 0 and retained radioactive nucleotide was quantitated with aMolecular Dynamics phosphorimager. Radioactive NTPs used were asindicated in figure and table legends. To evaluate rNTP/dNTPselectivity, reactions were run with 4 rNTPs and pT75 as template andα-P³² rNTPs or α-P³² dNTPs were used to label the transcripts. Therelative rate of incorporation of an rNTP vs. its cognate dNTP wasdetermined from the relative percentages of labeled rNTP vs. dNTPincorporated into DE81 retainable RNA at 5, 10, and 20 minute timepoints. Apparent miscoding frequencies were determined similarly, thoughin this instance the template was a single-stranded homopolymer and thereaction contained the complementary unlabeled rNTP, and complementaryα-P³² rNTP or one of the 3 non-complementary α-P³² rNTPs. The relativepercentages of labeled complementary vs. non-complementary rNTPsincorporated at 5, 10 and 20 minute time points gave an apparentmiscoding rate. The rNTP/dNTP selectivity assay was used to test thefollowing T7 RNAP mutants for effects on substrate discrimination:D537S, D537E, S539A, R551S, D552S, R627S, K631S, L637A, Y639S, Y639F,Y639A, G640A, F644A, G645A, Q649S, !810S, H811S, H811A, D812A, D812E,D812S, D812N, D879E+ΔF882+ΔA883, D879E+A881T+ΔA883, D879E+F884+A885,D879E+F882Y, D879E+ΔA883, D879E+F882W, D879E.

[0101] Elongation rate determinations were carried out as described(Golomb & Chamberlin, 1974) with some variations (Bonner, et al., 1994).

[0102] Determination of NTP K_(m) and k_(cat) was as describedpreviously (Patra, et al., 1992).

B. Results

[0103] Structure of the transcripts synthesized by Y639F and the w.t.enzyme with rNTPs and dNTPS: FIG. 1 diagrams the structure oftranscription products produced by Y639F and w.t. T7 RNAP in thepresence of various combinations of rNTPs and dNTPs. The template waspT75 (Tabor and Richardson, 1985) cut with HindIII so that transcriptionfrom its T7 promoter generates a 59-base run-off transcript.Electrophoresis was on a 20% polyacrylamide 6M urea gel. Plasmid andpolymerases were at concentrations of 10⁻⁷ M, and NTP concentrationswere 0.5 mM (all rNTPs and dTTP), 1 mM (dATP, dGTP), or 5 mM (dCTP).γ-P³²-GTP was added to radiolabel the transcription products. Wild-type(WT) or Y639F mutant (Mu) polymerases and NTPs used are as indicated.Poly-rG products of various sizes are labeled in lane A (“2G”, “3G”,etc. . . . ) and heterogeneous sequence abortive transcripts ofdifferent lengths are indicated by “4H”, “5H”, etc. . . . in lane c.Lanes q-t are a 10-fold longer exposure of lanes m-p.

[0104]FIG. 1 shows transcription reactions carried out with the w.t.enzyme or the Y639F mutant polymerase and a T7 φ-10 promoter template.Transcription by T7 RNA polymerase, like other RNA polymerases, ischaracterized by an initial, poorly processive ‘abortive’ phase oftranscription during which the short, nascent transcript frequentlydissociates from the ternary complex. When the transcript reaches alength of ˜9 bases transcription becomes highly processive and thetranscript becomes stably associated with the elongation complex. Onthis promoter, which initiates with GGGAGACCGGAAU (SEQ ID NO:1), T7 RNAPcan also synthesize long poly-G ladders (labeled “2G”, 3G“, etc. inlanes a and b) when rGTP is the sole NTP present (Martin, et al., 1988).Lanes c and d of the electrophoretic gel display the transcriptionproducts typical of runoff reactions counting all 4 rNTPs: there areabortive transcripts ranging up to 8 bases in length and a 59-baserunoff product. The 4 mer length the sequences of the poly-G transcriptsand the abortive transcripts made in the presence of all 4 rNTPs(labeled “4H”, “5H”, etc. ) diverge and no longer co-migrate with thepoly-G transcripts of equivalent size.

[0105] When rATP is omitted from the transcription reactions (lanes eand f) normal elongation of the initially synthesized GGG trimer cannotoccur. There are no heterogenous sequence abortive transcripts or 59base runoff products made and instead long poly-G transcript s, as inlane a, are made. Adding DATP to reactions lacking rATP does not changethe transcripts produced by the w.t. enzyme (lane g). However, with themutant enzyme we observed that addition of DATP (lane h) allowssynthesis a long runoff transcript as well as synthesis of heterogenoussequence abortive transcripts that do not co-migrate with the poly-Gtranscripts. Observation of an abortive transcript in lane h runningnear the position of the “4H” band in lane d confirms extension of theGGG trimer with an A but note that the major 4 mer transcript in lane hmigrates close to, but not precisely with, the major 4 mer in lane d orin adjacent lane i. This is consistent with the expectation that these 4mers will have identical sequence and length but different structure (i.e., rGrGrGrA in lanes d or i; rGrGrGdA in lane h). It should also benoted that some poly-G transcript synthesis is observed in lane h. Forexample, in lane h we observe both a heterogeneous sequence 4 mermigrating near the “4H” position and a smaller amount of 4 mer bandmigrating at the “4G” position. When 4 rNTPs are present (lanes c or d)the synthesis of poly-G transcripts is more completely suppressed. Thisindicates that dATP is utilized by Y639F, but not as efficiently asrATP.

[0106] When rCTP is omitted from the reaction, transcripts terminatepredominately at the 6 mer length because rCMP is normally firstincorporated at position 7 (lanes i, j). Addition of dCTP does not allowextension of the 6 mer in reactions with the w.t. enzyme (lane k).However, addition of dCTP to reactions with Y639F allows extensionbeyond the 6 mer length and synthesis of the runoff transcript (lane 1).Again, the following should be noted: 1. The transcripts larger than 6bases do not co-migrate with their counterparts in lanes c or dconsistent with the expected structural difference despite length andsequence identity, 2. there is more termination at the 6- and 7 merpoints in lane 1 than in lanes c or d, indicating that Y639F uses dCTPwell, but not as efficiently as it utilizes rCTP.

[0107] In lanes m and n UTP was omitted from the reactions. Lanes q-tshow a 10-fold longer exposure of lanes m-p. Within the set of 4 NTPs,UTP is unique on this template since it first becomes incorporated intothe transcript at the 13 base position. This corresponds to a transcriptlength subsequent to the transition form abortive to processivetranscription. As a consequence of this transition, the ternary complexbecomes more stable (Martin, et al., 1988). Therefore, when transcriptextension is blocked during the processive phase of transcription, thestalled ternary complex does not rapidly dissociate (Shi, et al., 1988).Instead it remains stalled on the template, near the promoter, andblocks reinitiation. For this reason we observe a large decrease in theoverall amount of transcription when UTP is omitted from the reaction inlanes m and n.

[0108] A longer exposure of lanes m and n (lanes q, r) does, however,reveal transcription products of the expected structure. When TTP isadded to these reactions (lanes o, p, s, t) synthesis of the 59 baserunoff transcript is observed with both the w.t. and Y639F mutant.

[0109] The ability of the w.t. enzyme to extend transcripts with TTPwhen it is unable to extend transcripts with the other dNTPs is alsolikely to be related to unique position at which UTP/TTP first becomesincorporated into the transcript. The amount of transcript terminationor extension that occurs with a particular dNTP depends simply on therelative rates of ternary complex dissociation or dNMP incorporation(McClure and Chow, 1980). During abortive transcription complex,dissociation must be more rapid than the rate at which the w.t. enzymeincorporates dNMPs into its transcripts. During processive transcriptioneven the expected slow rate of dNMP incorporation by the w.t. enzymemust be competitive with the slow rate of dissociation of the stableelongation complex, and an ability of the w.t. enzyme to incorporatedNMPs becomes manifest. Because elongation during the processive phaseof transcription is fast (˜230 bases/sec, Golomb and Chamberlin, 1974),while initiation and progression through abortive transcription is slow(Martin and Coleman, 1987; Martin, et al., 1988), elongation of thetranscript from 11 to 59 bases is expected to contribute less than 10%to the time required for transcript synthesis. As a consequence thephase of transcription during which TTP is incorporated may not be therate limiting step in synthesis of the runoff transcript even for thew.t. enzyme. These considerations imply that one need not expect to seemarked differences in synthesis of a relatively short runoff transcriptby Y639F or the w.t. enzyme when TTP is substituted for UTP.

[0110] Lanes u and v show reactions in which two rNTPs have beensubstituted with dNTPs (dATP and dCTP), and lanes w and x show reactionsin which three rNTPs have been substituted with dNTPs (dATP, dCTP, andTTP). Synthesis of the 59 base runoff transcript is observed with themutant polymerase (lanes v and x) indicating that Y639F can carry outsynthesis even when 3 rNTPs are substituted with dNTPs.

[0111] While the abortive transcript patterns in lanes v and x maylargely be described as a combination of the patterns observed in lanesh and 1 there are some important distinctions. The transcript patternsin lanes v and x reveal that the structure of the transcript, as well asthe structure of the NTP, affect the rate at which NMPs are added to thetranscript. For example, in lanes v and x there is an increase intermination at the 6 mer length relative to lane 1 (the 6 mer in lanes vor x runs slightly slower than the poly-G Smer in the adjacent lane). Inlanes 1, v and x synthesis of the 7 mer involves incorporation of dCMPbut in lanes v and x the 6 mer contains a dAMP at its 3′end and in lane1 it contains an rAMP.

[0112] The increase in termination at the 6 mer point in lanes v or xindicates that the presence of a dNMP on the 3′ end of the transcriptreduces the ability of the polymerase to further incorporate NMPs. Thehigh level of termination after synthesis of the rGrGrGdA 4 mer in laneh relative to the observed termination after synthesis of the rGrGrGrA 4mer in lanes c or d similarly indicates that the presence ofdeoxynucleotides in the transcript, at least at the 3′-end, influencessubsequent extension of the transcript.

[0113]FIG. 2 shows the effect of dGTP substitution on transcription bythe w.t. and Y639F polymerase.

[0114] Polymerases, template (supercoiled(Sc) or HindIII linearized (Li)pT75), and NTPs were as indicated. Polymerase and templateconcentrations and electrophoresis conditions as in FIG. 1. Left panel:Labeling was with α-P³² rGTP (a, d, g, j) or α-P³² dGTP(all otherlanes). The runoff transcript from the HindIII-cut template is indicatedin lane f.

[0115] Right panel: Labeling was with α-P³²-rGTP (a, b, f, g) orα-P³²-dGTP (all other lanes). Poly-rG and poly-dG products of varioussizes are indicated in lanes a, c, d. Alignment of these transcriptpatterns with those in lanes b, c, h, and i in the left panel revealsthat the added complexity of the transcript pattern in the latter set oflanes is due to the presence of a mixture of heterogeneous sequence andpoly-G transcripts. Heterogenous sequence abortive transcripts areindicated in lane c of the left panel (“4H”, “5H”).

[0116]FIG. 2 reveals the effects of substituting dGTP for rGTP intranscription reactions with the w.t. or Y639F polymerase. In reactionscontaining only dGTP and HindIII-cut pT75 as the template, the mutantpolymerase synthesizes poly-dG transcripts up to 4 bases in length (lanec, riqht panel). Note that—consistent with the assumed structuraldifferences—the poly-dG transcripts (“2dG”, “3dG”, etc. ) in lane c dono co-migrate with the poly-rG transcripts (“2rG”, “3rG”, etc. ) in lanea despite length and sequence identity.

[0117] When a supercoiled template is used, poly-dG transcripts up to 5bases in length are obtained (lane d, right panel). In lane e rGMP isadded to reactions which contain only dGTP.

[0118] Ribo-GMP can serve as the initiating, but not elongatingnucleotide during transcription( Martin and Coleman, 1989). With rGMP wetherefore ask whether either polymerase can elongate with dGTP if anrNMP is provided for initiation. Addition of rGMP to reactions with dGTPfurther extends the lengths of the transcripts obtained with the mutantpolymerase (lane e, right panel). With the w.t. enzyme very littlesynthesis is observed in reactions with dGTP (lanes h-j, right panel),though the normal pattern of poly-rG synthesis is observed in the rGTPreactions (lanes f, g, right panel).

[0119] When reactions contained three rNTPs and dGTP synthesis of runofftranscript from the HindIII-cut template is reduced much more than inreactions in which rGTP is present but other ribonucleotides aresubstituted with deoxynucleotides. For example, there is very littlerunoff transcript in lane h of the left panel of FIG. 2. Addition ofrGMP increases the amount of runoff transcript made by the mutant enzymein a reaction containing dGTP (lane i) but the amount of runofftranscript is still much less than in reactions with rGTP. On asupercoiled template (lanes a-c, left panel) high levels of longtranscripts are obtained with the mutant enzyme in the reactions withdGTP. The w.t. enzyme shows no transcript synthesis in any of thereactions with dGTP irrespective of whether supercoiled templates orrGMP is used.

[0120] Examination of FIG. 2 shows that the marked reduction in runofftranscript synthesis by the mutant enzyme in reactions with dGTP is notdue to a deficit in initiation. In fact, in all of the reactions withdGTP we observe abundant synthesis of 2-˜6 base transcripts with themutant enzyme. The low level of runoff transcript synthesis means thatthese short transcripts are being inefficiently extended to greaterlengths. It should also be noted that the abortive transcript patternseen in lanes b, c, h, and i of the left h panel in FIG. 2 is toocomplex to be accounted for by presuming one product of each baselength. Aligning the transcripts produced in the presence of dGTP onlywith those produced with dGTP, rUTP, rCTP, and rATP allows us toidentify the predominant abortive transcripts in lanes c, d, h, and i ofthe left panel as poly-dG products. In lane c of the left panel weindicate the major non poly-dG abortives by “4H” and “5H”. In lanes b,c, h, and i of the left panel of FIG. 2 we therefore see a patternsimilar to that of lane h in FIG. 1. The presence of a mixture ofnormally extended heterogenous sequence and poly-G abortive transcriptsindicates that ′normal′ transcript extension is inefficient.

[0121] It has been shown that mutations in T7 RNAP that reducephosphodiester bond formation rates cause poly-rG transcript synthesiseven when 4 rNTPs are present (Bonner, et al., 1994). It can be seenthat the ratio of poly-G to heterogenous sequence transcripts in lanesb, c, h, i of the left panel of FIG. 2 is greater than in lane h of FIG.1, indicating a greater deficiency in normal transcript extension whendGTP is substituted for rGTP than when dATP is substituted for dGTP.Note that this occurs even though normal extension of the dGdGdG trimerin lane i of the left panel of FIG. 2 (for example) would involveaddition of a ribo-AMP while extension of the rGrGrG trimer in lane h ofFIG. 1 would involve addition of a deoxy-AMP, clearly highlighting therole of transcript structure, as well as substrate structure, indetermining the efficiency of transcript extension. These results showthat Y639F can initiate and elongate transcripts with dGTP substitutedfor rGTP but normal extension of the transcripts in the 2-8 base rangeis impaired leading to a large increase in the proportion of poly-dG andshort transcripts synthesized. Addition of rGMP to serve as theinitiating nucleotide and the use of a supercoiled template both enhancethe ability of the mutant to extend the short transcripts during theinitial stages of transcription.

[0122] Barriers to initiation and extension of the initial transcriptwith dNTPs: FIG. 2 reveals that Y639F can efficiently transcribe withdGTP the initial G segment of a promoter that initiates “GGGA” but isseverely blocked in extending the dG trimer with the A. This impliesthat the sequence of the initially transcribed region may influence theefficiency with which Y639F can extend the transcript when using dNTPs.FIG. 2 also shows that supercooling, which presumably facilitatesunwinding of the template, enhances the activity of Y639F when usingdNTPs. To evaluate the effects of sequence and single-strandedness inthe initially transcribed region on the activity of Y639F when usingdNTPs, we examined transcription from a set of synthetic promoters whichdiffered in the sequence of their initially transcribed regions and inbeing fully double-stranded or single-stranded in their initiallytranscribed regions (FIG. 3).

[0123]FIG. 3 shows the effects of single-strandedness and sequence inthe initially transcribed region on the activity of Y639F in reactionswith 4 rNTPs or 4 dNTPs. Poly-rG transcripts of various sizes areindicated in lane a. Reactions contained the indicated NTPs andpolymerases. Polymerase and promoter concentrations were 10⁻⁶ M and 10⁻⁵M, respectively. NTP concentrations and electrophoresis as in FIG. 1.Indicated in some of the lanes are the sequences of differenttranscripts as deduced from alignment with poly-rG or poly-dG ladderssynthesized in the presence of rGTP or dGTP only. The synthetic promotertemplates used are double-stranded and have thesequence—CGAAATTAATACGACTCACTATA (SEQ ID NO:2)—in their -23 to -1regions. The promoters differ in their initially transcribed regions asfollows: b-e, t, u: GGACT; f-j, v, w: GAGACCGG; a, j-m, x, y: GGGAGACC;n, o, z: GGAAAATT; p-s: GGGGGGGGGGGACT (SEQ ID NO:3). The promoters alsodiffer in being double-stranded (b, d, f, h, j, 1, p, r) orsingle-stranded (other lanes) in their transcribed regions.

[0124] For most of the promoters tested, transcription with rNTPs wasnot markedly affected by having the initially transcribed region besingle-stranded. However, when transcribing with 4 dNTPs, Y639F was moreactive on the partially single-stranded promoters. For example, in lanee (partially single-stranded promoter) of FIG. 3 transcription productsare both more abundant and extend to greater lengths than in lane d,where the promoter is fully double-stranded. A similar comparison may bemade between lanes m and 1 or s and r. Regarding sequence we found thata promoter which initiated with 3 G's was superior to a promoter whichinitiated with two, which was in turn superior to a promoter whichinitiated with just one G. Thus Y639F activity when using dNTPs wasgreatest on a promoter which initiates “GGGAGACC” (lanes 1 and m). Theinitially transcribed region of this promoter corresponds to theconsensus sequence for T7 promoters in the +1 to +6 segment. This wasthe only promoter which, when fully double-stranded, gave rise to highlevels of transcript synthesis with Y639F in reactions containing onlydNTPs (lane l).

[0125] Promoters which initiated with 2 G's (lanes d, e, o) gave lowerlevels of transcript synthesis in the 4 dNTP reactions, and a promoterwhich initiated with 1 G (lanes h, i) was not utilized by Y639F inreactions with 4 dNTPs. Within the initially transcribed region,elements other than the number of G's appear to be important. Forexample, we have found that the w.t. or Y639F polymerases are lessefficient in initial transcription extension on the T7 promoter found inthe pBS plasmid which initiates GGGC, than on the φ10 promoter found inpT75 which initiates with the consensus GGGAGA (data not shown).

[0126] Another example is evident in lanes n and o which show thetranscripts obtained on a partially single-stranded promoter thatinitiates GGAAAAUU. Like the promoter used in lanes c and e, thispromoter initiates with 2 G's but normal transcript extension on thispromoter is less efficient than on the promoter that initiates GGACU. Inthe 4 rNTP reactions (lanes c, n), the proportion of short transcripts(dimers, trimers) is greater in lane n and we observe significantamounts of poly-rG transcripts beyond the dimer length in lane n but notin lane c. In the 4 dNTP reactions almost all of the transcripts in laneo terminate at the trimer or tetramer length. Because increasing thenumber of Gs from 1 to 3 enhanced Y639F activity when using dNTPs, wetested a promoter which initiated with a run of 11 G's (lanes p-s). Apotential drawback to such a promoter is that such a long run of G'scould inhibit the ability of the polymerase to unwind the template.Since T7 promoters with more than 3 consecutive G's in the initialregion do not occur naturally, it may be that for other reasons suchsequences do not favor initial transcript extension. In fact, we findthat this promoter is a poor template. When it is fully double-stranded,initiation and extension of the transcript is inefficient with eitherrNTPs (lane p) or dNTPs (lane r), consistent with the expectation that apromoter with this sequence would be difficult to melt. Initiation andtranscript extension is enhanced when this promoter is partiallysingle-stranded (lanes q and s), but while poly-G transcripts from 2 to7 or more bases in length are abundant, runoff transcripts of theexpected length are not predominant products. In reactions with 4 rNTPsthe transcript patterns of the w.t. and Y639F polymerases are virtuallyidentical so we do not repeat the 4 rNTP reactions with the w.t. enzymein FIG. 3. Lanes t-z show that the w.t. enzyme is virtually inactive inreactions with 4 dNTPs and the same set of promoter used in lanes b-s.

[0127] Relative selectivity of the mutant and w.t. polymerases for dNTPsand rNTPs: FIGS. 1-3 present a qualitative analysis of the structure ofthe transcripts produced by the w.t. and Y639F polymerases with variouscombinations of NTPs. They show that Y639F can use dNTPs with highefficiency and that both transcript and substrate structure play a rolein determining the efficiency of transcript extension. To obtain aquantitative measure of the relative selectivity of the w.t. and mutantpolymerases for dNTPs vs. rNTPs under conditions where transcriptstructure was not a complicating factor, we carried out reactions inwhich all 4 rNTPs were present but rNTPs or dNTPs were used toradiolabel the transcripts (Table I, see Appendix 1). Under theseconditions the unlabeled rNTPs were present in vast excess relative tothe labeling NTPs so labeling dNTPs are almost always incorporatedadjacent to rNTPs and into transcripts of nearly uniform rNMP structure.On average the mutant enzyme is ˜20-fold less selective for rNTPs overdNTPs than the w.t. enzyme. We used this assay to screen our collectionof T7 RNAP active site mutants for increased dNTP utilization. In thisscreen we looked for increased dATP incorporation in transcriptionreactions with all of these mutants using supercoiled pT75 as thetemplate, 4 cold rNTPs, and P³²-rATP or P³²-DATP to label. Since theseresults were negative with the exception of the tyrosine 639 mutants, wedo not present them here, but the mutants tested in this way are listedin “Materials and Methods”. It has been reported that use of Mn++instead of Mg++ decreases substrate discrimination and increasesmiscoding for a number of polymerases (Tabor and Richardson, 1989;Nivogi and Feldman, 1981). We, therefore, examined the rNTP/dNTPselectivity of the mutant and w.t. enzymes in Mn++ -citrate buffer(Tabor and Richardson, 1989). With Mn++ the preference of both the w.t.and Y639F polymerases for rNTPs over dNTPs was markedly reduced.

[0128] Relative activity of the w.t. and Y639F polymerases withdifferent NTP combinations: The relative activities of the Y639F andw.t. polymerases with supercoiled pT75 as a template and variouscombinations of rNTPs/dNTPs were measured in both Mg++ and Mn++ buffers(Table II, see Appendix 1). In Mg++ buffer, substitution of a singlerNTP with a dNTP reduces w.t. activity by 20 to >400-fold, but onlymodestly reduces the activity of Y639F. The rank order of the effect ofa particular dNTP substitution on w.t. enzymeactivity—dGTP>dATP>dCTP>dTTP=dUTP—matches the order of their addition tothe transcript. With the “2 dNTP” reactions the w.t. enzyme was mostactive when rCTP and rUTP were substituted with dNTPs, corresponding tothe 2 nucleotides added latest to the transcript. The w.t. enzyme wasinactive with all other “2 dNTP” or “3 dNTP”. In the “2 dNTP” reactionsY639F was least active in the dGTP, DATP reaction, corresponding to the2 nucleotides incorporated first during transcription. In the “3 dNTP”reactions Y639F was least active in the dGTP, dATP, dCTP reaction,corresponding to the 3 nucleotides incorporated first duringtranscription.

[0129] In Mn++ buffer both the w.t. enzyme and Y639F show a reduction intheir sensitivity to substitution of dNTPs for rNTPs, consistent with anexpectation of reduced substrate discrimination in Mn++ buffer. Therewas, however, also a sharp reduction in overall activity with Mn++. Wevaried Mn++ concentrations over a wide range (from 20 mM to 150 μM in2-fold dilutions) to determine if an optimal Mn++ concentration thatwould result in high activity could be identified, but we found similaractivity at all Mn++ concentrations tested (data not shown). Thus, whilediscrimination between rNTPs and dNTPs was less in Mn++ buffer, Y639F ismore active in Mg++ buffer than in Mn++ buffer with all NTP combinationsexamined. Similarly, the w.t. enzyme exhibits greatly reduceddiscrimination between rNTPs and dNTPs in Mn++ buffer, but is modestlymore active in Mn++ buffer than in Mg++ buffer only for certaincombinations of dNTPs and rNTPs.

[0130] DNA and RNA synthesis on homopolymeric templates: T7 RNAP willsynthesize poly(rG) RNAs on poly(dC) templates (Bonner, et al., 1994;Ikeda and Richardson, 1987). We measured the activity of the w.t. andmutant polymerases on poly(dI)-poly(dC) with rGTP, dGTP, dGTP+rGMP(Table III, see Appendix 1). The activity of T7 RNAP onpoly(dI)-poly(dC) and poly(dC) is especially robust. Mutant polymerasesthat have greatly reduced activity on normal promoter templates stilldisplay high activity on poly(dC) templates (Bonner, et al., 1994). We,therefore, characterized two poorly active non-conservative tyrosinemutations on this template (Y639A and Y639S). In Table III we alsopresent results obtained with mutant G640A. Presentation of data for thelatter mutant was selected because it is more comparable in activity tothe Y639A/S mutants and because it is representative of a mutation whichhas marked effects on the kinetics of transcription but does not affectsubstrate discrimination, even though it is directly adjacent to Y639.We find that all of the Y639 mutants exhibit reduced substratediscrimination as demonstrated by the fact that their differentialactivity in reactions containing dGTP or dGTP+rGMP vs. rGTP is less thanfor the w.t. enzyme or the G640A mutant. In fact Y639F displays similaractivity with rGTP, dGTP, or dGTP+rGMP.

[0131] Since the nucleic acid synthesized by Y639F on poly(dI)·poly(dC)using dGTP is presumably composed solely of dNMPs it is expected to beresistant to alkaline hydrolysis (Schmidt and Tannhauser, 1945). FIG. 4shows transcription by Y639F and w.t. polymerase with dGTP or rGTP onpoly(dI)·poly(dC). Transcription reactions were carried out withpoly(dI)·poly(dC) at 0.2 mg/ml and the indicated NTPs and polymerases.Reaction products were left untreated (−) or treated with 1 M NaOH for 5hours at 37° C. (+). Polymerase and NTP concentrations andelectrophoresis as in FIG. 1. Labeling was with α-P³² rGTP (a, d, g, j)or α-P32 dGTP (other lanes). FIG. 4 shows the transcription productsobtained with the w.t. or Y639F polymerases on poly(dI)·poly(dC) beforeand after treatment with alkali. With dGTP or dGTP+rGMP the w.t. enzymeis poorly active in the synthesis of long transcripts, however we canobserve smears of heterogeneously sized, short transcripts in thereactions with the w.t. enzyme and the dGTP substrates (lanes e and f)while Y639F synthesizes higher levels of long transcripts which areretained near the top of these gels (lanes b and c). The presence ofthese short transcripts indicates that Y639F and the w.t. enzyme differonly in the degree to which they can utilize dNTPs. The w.t. can alsoinitiate and extend transcripts with dGTP, but it is much lessprocessive when using dNTPs than Y639F so its transcripts are muchshorter. When these reactions are treated with base, degradation of thelong transcripts made in the reactions with rGTP is observed and theamounts of short RNAs (presumably hydrolysis products) increase (lanes gand j). In the reactions in which dGTP or dGTP+rGMP were used assubstrates no degradation of the transcripts by base treatment isobserved, confirming that these transcripts are composed of dNMPs.

[0132] T7 RNAP as a reverse transcriptase or RNA replicase whichinitiates de novo: It has been reported that T7 RNAP can use both RNAand DNA templates (Konarska and Sharp, 1989). We, therefore, determinedif the Y639F mutant would use dNTPs when transcribing an RNA template(poly(rC), Table IV). Overall the activity of the w.t. and Y639Fpolymerases on poly(rC) with rGTP was 10-20-fold less than on poly(dC)(not shown), but this reduction did not preclude synthesis of highlevels of RNA on poly(rC) by using higher polymerase concentrations thanwere used in the poly(dI)·poly(dC) reactions. When dGTP or dGTP+rGMP wasused the w.t. enzyme was not measurably active on poly(rC), while theactivity of Y639F was reduced by only ˜4-fold (with dGTP+rGMP) or˜8-fold (with dGTP). Thus, both the w.t. and Y639F polymerases arecapable of unprimed RNA-directed RNA polymerization while Y639F is alsocapable of unprimed reverse transcription.

[0133] DNA- and RNA-primed synthesis of DNA and RNA: In the assaysdescribed so far we have examined de novo initiated synthesis. T7 RNAPcan also extend RNA primers. We, therefore, examined the abilities ofboth polymerase to carry out DNA or RNA primed synthesis of DNA and RNA(FIG. 5).

[0134]FIG. 5 shows primed synthesis of DNA and RNA with Y639F and thew.t. polymerase. Transcription reactions contained end-labeled 12 baseDNA or RNA primers of identical sequence (GGACACGGCGAA, SEQ ID NO: 4)hybridized to a DNA template(CCCGGGATGGAATGGAGTATTCGCCGTGTCCATGGCTGTAAGTATCC, SEQ ID NO: 5).Primer-template concentration was 10⁻⁵ M. Reactions contained theindicated polymerases (10⁻⁷ M) and NTPs. NTP concentrations andelectrophoresis as in FIG. 1.

[0135] We found that both the w.t. and Y639F polymerases can extend DNAand RNA primers with rNTPs, but extension of DNA primers was 2-3-foldless efficient than extension of RNA primers. Y639F also extended DNAand RNA primers with dNTPs but ˜4-fold less efficiently than with rNTPs.

[0136] The Y639F mutant does not exhibit greatly increased miscoding: Weexamined the miscoding properties of the w.t. and mutant T7 RNAPs bymeasuring the relative incorporation of labeled rGTP, rUTP, rATP, andrCTP on poly(dC) or poly(dT) templates in the presence of excessunlabeled rGTP or rATP, respectively (Table V, see Appendix 1). Anincrease in miscoding would be reflected in an increase in the rate ofincorporation of the non-complementary NTP into RNA.

[0137] On poly(dC), the w.t., Y639F, and G640A polymerases incorporaterGTP into RNA at greater than 1300-2000-fold the rate of rUTPincorporation. Ribo-GTP is incorporated some 400-600-fold better thanrCTP, and 200-400-fold better than rATP. Because of their lower activitywe can say only that the relative rate of incorporation of rGTP onpoly(dc) is 184-fold greater than the rate of incorporation ofnon-complementary rNTPs for Y639A, and 50-fold greater for Y639S. Theuse of Mn++ instead of Mg++ has been reported to increase miscoding fora number of polymerases (Tabor and Richardson, 1989; Nivogi and Feldman,1981) so we examined the effects of Mn++ on miscoding by w.t. and Y639Fpolymerases. In Mn++ buffer the G640A, Y639A, and Y639S mutants wereinsufficiently active to allow accurate measures of miscoding. With thew.t. and Y639F polymerases the use of Mn++ increases miscoding by20-40-fold. However, the apparent rate of miscoding by Y639F remainssimilar to the w.t. enzyme.

[0138] On poly(dT), high levels of activity allowing an accurate measureof miscoding frequencies could only be observed for Y639F and the w.t.polymerase in both Mg++ and Mn++ buffers. In Mg++ buffer apparentmiscoding rates on poly(dT) were higher than on poly(dC), but weresimilar for Y639F and the w.t. enzyme. In Mn++ buffer miscoding rateswere increased by ˜5-fold, on average, but again rates were similar forw.t. and Y639F. However, on poly(dT) Y639F did show a reproducible˜2-fold increase, relative to the w.t. enzyme, in the ratio of the ratesof rGTP to rATP incorporation.

[0139] Homopolymer assays have been used previously to measure miscodingby RNAPs (Nivogi and. Feldman, 1981; Glazer, 1978; Blank, et al., 1986)but it should be remarked that they can produce only upper bounds formiscoding frequencies. Measured miscoding rates could reflectcontamination of the homopolymeric templates. It is also possible thatthe transcripts themselves could serve as templates and supportincorporation of rNMPs non-complementary to the original template (i.e.,the poly(rG) or poly(rA) transcripts made on poly(dC) or poly(dT) couldsubsequently support synthesis of poly(rC) or poly(rU) transcripts).Such caveats are less relevant to the miscoding observed in Mn++ buffersince the change in divalent cation increases miscoding but cannotaffect template composition. However, in Mg++ buffer we should considerthe measured miscoding frequencies to be upper bounds for the true ratesof miscoding. Nevertheless, the results presented in table V indicatethat Y639F does not exhibit a gross increase in miscoding which wouldmanifest itself as a clear increase in the incorporation ofnon-complementary rNMPs on homopolymeric templates.

[0140] The increased utilization of dNTPs by Y639F is due to both adecreased K_(m) and an increased k_(cat) for dNTPs: The K_(m) andk_(cat) of the w.t. and Y639F polymerases with rATP, rITP and 5different dNTPs were measured (Table VI, see Appendix 1). The K_(m) ofthe w.t. enzyme for dNTPs was much higher than previously reportedvalues for the corresponding rNTPs and varied considerably for differentdNTPs (Ikeda and Richardson, 1987; Patra, et al., 1992). Notably thew.t. enzyme K_(m) values correlate with the rNTP/dNTP selectivity valuespresented in Table I. The selectivity of the w.t. enzyme with ribo- vs.deoxy-nucleotides was greatest for CTP, followed by ATP, and was theleast for UTP, implying that an important component of the selectivityof the w.t. enzyme for rNTPs over dNTPs is a much higher K_(m) fordNTPs. For Y639F, the K_(m) values for these dNTPs are from ˜3 to˜11-fold less, but the rank order of these K_(m) values (dCTP K_(m)>dATPK_(m)>dGTP K_(m)>dTTP K_(m)) is the same as for the w.t. enzyme. ForY639F, k_(cat) values for reactions with different dNTPs were only 2-4fold less than for rNTPs, while the w.t. enzyme displayed k_(cat) valueswith dNTPs that were from ˜6 to ˜30-fold less than for rNTPs.

[0141] Elongation rates of Y639F in reactions containing a single dNTP:Elongation rates for Y639F in reactions with 4 rNTPs or 1 dNTP and 3rNTPs were determined by analyzing aliquots, taken at 10 secondintervals, from transcription reactions initiated on supercoiled PT75 byadding NTPs to otherwise complete reaction mixes (FIG. 6).

[0142]FIG. 6 shows relative elongation rates of Y639F in “4 rNTP” and “3rNTP+1 dNTP” reactions. The template was supercoiled pT75 at 5×10⁻⁷ M.Y639F polymerase was used at a concentration of 10⁻⁶ M. Reactionscontained the indicated NTPs. Labeling was with α-P³²-rATP (lane e) orα-P³²-rGTP (other lanes). After initiation of the reactions aliquotswere taken at 10 second intervals and analyzed on 1% agarosedenaturing-formaldehyde gels. The figure shows the 20 second time point.The bars indicate the positions of λ DNA markers.

[0143] When analyzed on denaturing agarose gels (FIG. 6) theheterogeneously sized transcripts from these reactions are resolved as asmear with the trailing edge of the smear corresponding to transcriptsinitiated at t=0 and from which the maximal transcript elongation ratecan be determined (Golomb and Chamberlin, 1974; Bonner, et al., 1994).The following elongation rate reductions (relative to a ‘4 rNTP’reaction) were obtained for reactions containing a 3 rNTPS and 1dNTP:dATP, ˜3-fold; dUTP, ˜2-fold; dGTP ˜1. 5-fold; dCTP, 1-1.5-fold.Because of its poor activity in reactions with dNTPs we could notdetermine the corresponding elongation rate reductions for the w.t.enzyme.

[0144] Other non-canonical nucleoside triphosphate substrates for mutantpolymerases: Although wild-type T7 RNAP can not efficiently utilizedideoxy-NTPs as substrates, we have found that Y639 mutants of thisenzyme can also use dideoxy-NTPs as substrates (Table VII). We have alsofound that Y639 mutants can use other non-canonical nucleosidetriphosphates as substrates (Table VIII). Nucleic acids synthesized byincorporation of some non-canonical nucleotides, such as 2′-F-NTPs, mayoffer advantages in being more resistant to digestion by nucleases suchas ribonucleases. Other uses and advantages of various non-canonicalnucleotide substrates in methods of the present invention using themutant polymerases will be apparent after examination of thespecification, claims and drawings.

[0145] Other mutations: It is conceivable that, in the absence of abound rNTP, a hydrogen bond forms between Y639 and some other activesite side chain. The possibility of an interaction between M635 and Y639was tested since M635 and Y639 are close and M635 approaches the ribosein our models of NTP in T7 RNAP (Huang, et al., submitted forpublication). This position is methionine in the T7 RNAP class of RNAPs(McAllister, W. T., 1993), but is either tyrosine or phenylalanine inthe homologous DNAPs, and in the DNAP mutants at this site (i.e.,positions homologous to position 762 in E. coli DNAP I) that affectdNTP/ddNTP discrimination (Tabor. S., and Richardson, C. C., Europeanpatent application, 1994). While M635A, M635F or M635Y mutants hadeffects on NTP K_(m), they did not affect 2′-group discrimination withrespect to dNTPs and rNTPs in either wild-type or the Y639F T7 RNAPbackground. Additional studies will reveal whether these M635 mutationshave other effects, such as effects on discrimination at the 3′-positionof the sugar, or effects on discrimination with respect to NTPs withother substituents, such as fluorine at the 2′ position of the sugar.Also, studies on similar double mutations at the homologous sites in thehomologous class I DNAPs will reveal the effects of such doublemutations on discrimination at the 2′- and 3′-positions of the sugar;specifically, these studies will reveal whether class I DNAPs having aphenylalanine at the position homologous to amino acid position 766 inE. coli DNAP I have reduced discrimination for rNTPs if the amino acidis methionine or tyrosine at the position homologous to amino acidposition 762 in E. coli DNAP I.

[0146] Provided that class I DNAP mutants which have a reduceddiscrimination for rNTPs compared to dNTPs can be obtained, it will bepossible to use such DNAP mutants to carry out the methods of thepresent invention that comprise nucleic acid synthesis from a nucleicacid primer, at least part of which is sufficiently complementary to atemplate nucleic acid to hybridize therewith and to be extended by thepolymerase. An especially preferable use for such mutant DNA polymeraseswould be to carry out Partial Ribo-substitution sequencing reactionsfrom primers, whether labelled by any of the methods known in the art,or unlabelled. Since the sequence-delimiting rNTP nucleotides for thePartial Ribo-substitution Reaction do not terminate the growingphosphodiester chain when they are incorporated during nucleic acidsynthesis, the DNA synthesis for Partial Ribo-substitution can occursimultaneous with and be identical to nucleic acid synthesis for anotherprocedure, such as NASBA, 3SR, TMA or another similar method, providedthat the mutant DNAP with reduced discrimination for rNTP compared todNTPs is thermostable, or the PCR strand displacement amplification orother methods for nucleic acid amplification involving nucleic acidsynthesis from a primer. The nucleic acid products containing thesequence-delimiting rNMPs can then be cleaved by treatment with achemical base or a ribonuclease, and analyzed by methods known in theart to obtain the nucleotide-specific pattern of bands or, provided thata Partial Ribo-substitution Reaction is carried out for each of the fournucleotides, the complete sequence of the nucleic acid. Also, providedthat primers with distinguishable non-radioactive labels are used,multiple Partial Ribo-substitution sequencing reactions may be carriedout simultaneously in the same reaction mixture and run and read in thesame lane of a polyacrylamide gel or capillary tube or other matrix forseparating the fragments based on size, as is the case for all of themethods of the present invention described herein.

C. Discussion

[0147] Our results reveal that mutations of tyrosine 639 in T7 RNAPreduce the ability of the polymerase to discriminate between rNTPs anddNTPs. A conservative mutation which removes the tyrdsine hydroxyl butretains the phenolic ring (Y639F) exhibits w.t. activity but an averagereduction of ˜20-fold in the selectivity for dNTPs over rNTPs (Table I).Non-conservative mutations of this tyrosine (Y639A/S) also displaydecreased rNTP/dNTP discrimination (Table III), but are less active thanthe w.t. enzyme. Replacement of an rNTP by a dNTP typically reducesY639F transcript elongation rates by only a factor of two. Tyrosine 639is conserved in a large number of DNA-directed RNA and DNA polymerases(Delarue, et al., 1990). In DNAP I, mutations of the Y766 to serine andphenylalanine have been characterized (Polesky, et al., 1989; Carrol, etal., 1991). The Y766S mutation was alone amongst a number of active sitemutations characterized in decreasing DNAP I fidelity (increasingmiscoding). The Y766F mutation displayed w.t. fidelity and activity.Similarly, the T7 RNAP Y639F mutant displays w.t. kinetics (Bonner, etal., 1992, 1994; Woody, et al., 1994), and the only effect we canidentify for this mutation in T7 RNAP is the reduced substratediscrimination reported here. Thus, while T7 RNAP Y639F showed decreaseddNTP/rNTP selectivity, it did not exhibit increased miscoding asassessed by incorporation of non-complementary NTPs on homopolymerictemplates.

[0148] The Y639 T7 RNAP mutations present us with, in one sense, thefunctional unification of polymerases to go along with their structuralunification. The active site of w.t. T7 RNAP is forgiving with regardsto template structure (RNA or DNA) or mode of initiation (primed or denovo). A mutation which relaxes the substrate selectivity of thispolymerase further expands the range of activities which it can displayin vitro. Depending on the substrates and templates presented to it, theY639F T7 RNAP can act as an RNA- or DNA-directed RNA or DNA polymerasein primed or de novo initiated reactions. Thus it can display a varietyof activities normally associated with distinct polymerases, includingsome entirely novel activities such as de novo initiated reversetranscription or mixed dNMP/rNMP polymer synthesis.

Example 2: A mutant SP6 RNA Polymerase as a DNA Polymerase

[0149] After the observations made above with T7 RNAP, we decided toexamine bacteriophage SP6 RNA polymerase to determine whether the DNAsynthesis properties observed for the mutant T7 RNAP could, as expected,be extended to other mutant polymerases. Bacteriophage SP6 is a lyticphage which infects the bacterial species Salmonella tryphimurium(Butler and Chamberlin, 1982). SP6 phage resembles E. coli phage T7 andtheir genomes are comparable in size, gene organization and pattern ofgene expression (Kassavetis, et al., 1982).

[0150] The phage encoded RNA polymerases are very similar in size(Butler and Chamberlin, 1982) and amino acid sequence (Katani, et al.,1987).

[0151] The homologous tyrosine at position 639 in T7 RNA polymerase isreadily identified at position 631 in SP6 RNA polymerase (FIG. 7).Substitution of tyrosine 631 with phenylalanine in the SP6 RNApolymerase was expected to confer the same phenotypic changes incatalytic properties in this enzyme as were demonstrated for Y639FT7 RNApolymerase (Example 1).

[0152] Localized Mutagenesis. Refer to FIG. 7 for a summary of the aminoacid and nucleotide sequence surrounding TYR631 in the SP6 RNApolymerase gene. Mutagenesis of the Y631 residue may be accomplished bythe method of Kunkel, et al. (1991). Alternatively, one of the manyother methods for mutagenesis known to those of skill in the art may beused. The amino acid and nucleotide sequences of the resulting TYR631PHEmutant SP6 RNA polymerase are also given in FIG. 7. As shown, the Aresidue at position 2 in codon 631 of the SP6 RNA polymerase gene waschanged to a T. This results in the loss of the single NdeI restrictionenzyme site which is present in the wild-type gene, permittingidentification of mutant clones.

[0153] Preparation and Purification of Mutant RNA Polymerase

[0154] A single clone was selected in which the NdeI site was missingand which expressed SP6 RNA polymerase activity. Several liters ofLB+Amp were grown from a pUC18 Y631F SP6 RNA polymerase clone overnightat 37° C. Cells were harvested, lysed and Y631F mutant SP6 RNApolymerase was purified approximately according to standard methods(Butler and Chamberlin, 1983).

[0155] Transcription Reactions

[0156] To verify that Y631F SP6 RNAP had the desired phenotype, in vitrotranscription reactions were done where one of the four rNTPs wassubstituted by the corresponding dNTP (Sousa and Padilla, 1995). Asexpected, the Y631F SP6 RNAP mutant displayed reduced dNTP/rNTPdiscrimination compared with wild-type SP6 RNAP, similar to thatobserved for the Y639 mutant of T7 RNAP.

[0157] In a standard in vitro transcription reaction using the fourribonucleoside triphosphates (rATP, rGTP, rCTP, and rUTP), both enzymes,the wild-type SP6 RNA polymerase as well as the Y631F mutant,synthesized the correct 1.4 kb transcript and in the expected amounts asvisualized on gels. However, if one of the four ribonucleosidetriphosphates, rGTP for example, is completely substituted by dGTP andin vitro transcription reactions are done with the wild-type and mutantenzymes, no transcript is made by the wild-type enzymes. However, themutant enzyme makes the expected full length transcript in good yield asobserved on agarose gels.

1 5 1 13 RNA Artificial Sequence Description of Artificial SequenceRegion of T7 promoter 1 gggagaccgg aau 13 2 23 DNA Artificial SequenceDescription of Artificial Sequence Portion of synthetic promotertemplate 2 cgaaattaat acgactcact ata 23 3 14 DNA Artificial SequenceDescription of Artificial Sequence Portion of synthetic promotertemplate 3 gggggggggg gact 14 4 12 DNA Artificial Sequence Descriptionof Artificial Sequence Oligonucleotide primer 4 ggacacggcg aa 12 5 47DNA Artificial Sequence Description of Artificial SequenceOligonucleotide primer 5 cccgggatgg aatggagtat tcgccgtgtc catggctgtaagtatcc 47

We claim:
 1. A method for synthesizing a nucleic acid moleculecomprising at least one non-canonical nucleotide, comprising the stepsof: a) incubating a template nucleic acid in a reaction mixture undernucleic acid synthesis conditions containing (i) a mutant nucleic acidpolymerase, wherein said polymerase has a reduced discrimination betweencanonical and non-canonical nucleoside triphosphates, and (ii) at leastone non-canonical nucleoside triphosphate, wherein said non-canonicalnucleoside triphosphate is incorporated into the synthesized nucleicacid in place of only one canonical nucleoside triphosphate, and b)obtaining the synthesis of a nucleic acid molecule comprising at leastone non-canonical nucleotide.
 2. The method of claim 1 wherein thetemplate nucleic acid is DNA.
 3. The method of claim 1 wherein thetemplate nucleic acid is RNA.
 4. The method of claim 1 wherein a nucleicacid molecule comprising at least one non-canonical nucleotide issynthesized by extension of a primer molecule, at least part of which issufficiently complementary to a portion of the template to hybridizetherewith.
 5. The method of claim 1 wherein a nucleic acid moleculecomprising at least one non-canonical nucleotide is synthesized de novowithout using a primer molecule.
 6. The method of claim 1 wherein thepolymerase is an RNA polymerase.
 7. The method of claim 1 wherein thepolymerase is a T7-type RNA polymerase.
 8. The method of claim 1 whereinthe polymerase is selected from the group consisting of T7 and SP6 RNApolymerases.
 9. The method of claim 1 wherein the mutant polymerase isan RNA polymerase and the non-canonical nucleoside triphosphate is a2′-fluoro-nucleoside triphosphate.
 10. The method of claim 1 wherein thesynthesized nucleic acid molecule has an altered susceptibility to aribonuclease or a deoxyribonuclease compared to a nucleic acid which issynthesized using the corresponding non-mutant nucleic acid polymerase.11. The method of claim 1 wherein the synthesized nucleic acid moleculeis selected from the group consisting of a ribozyme or a nucleic acidmolecule used for gene therapy, in a vaccine, in an antiviralcomposition, in an antimicrobial composition, in an antisensecomposition for regulating gene expression, in a composition forhybridization to a complementary nucleic acid, or as a probe fordetection of a complementary nucleic acid.
 12. The method of claim 1wherein the synthesized nucleic acid molecule is single-stranded.
 13. Akit for performing the method of claim 1, comprising a mutant nucleicacid polymerase which has reduced discrimination between canonical andnon-canonical nucleoside triphosphates and data or informationdescribing conditions under which the method of claim 1 may beperformed.
 14. The kit of claim 13, wherein the nucleic acid polymeraseis a mutant T7-type RNA polymerase.
 15. The kit of claim 13, wherein thenucleic acid polymerase is a T7 RNA polymerase comprising an alteredamino acid at position
 639. 16. The kit of claim 13, wherein the nucleicacid polymerase is SP6 RNA polymerase comprising an altered amino acidat position
 631. 17. A method for determining the sequence of a nucleicacid molecule using a mutant RNA polymerase which has a reduceddiscrimination for non-canonical versus canonical nucleotides assubstrates, comprising the steps of: a) synthesizing a nucleic acidmolecule de novo from an RNAP promoter sequence in a reaction mixturecontaining the mutant RNA polymerase in each of four separate reactions,each reaction comprising at least four nucleoside triphosphates, whereinat least one nucleoside triphosphate has a nucleic acid base which. iscomplementary to each of adenine, cytidine, guanine and uracil orthymine and a sugar with either a hydroxy or a hydrogen or a fluorine atthe 2′-position, and a portion of a ddNTP, such that each of the fourseparate reactions contains a ddNTP that is complementary to a differentone of the four common nucleic acid bases in a nucleic acid molecule,and b) evaluating the reaction products so that the sequence of thetemplate molecule may be deduced.
 18. The method of claim 17 wherein,each reaction comprises at least four nucleoside triphosphates chosenfrom the group consisting of ATP, CTP, GTP, and UTP or rTTP, and furthercomprises one ddNTP.
 19. The method of claim 17 wherein, each reactioncomprises at least four nucleoside triphosphates chosen from the groupconsisting of dATP, dCTP, dGTP, dUTP, dTTP, 7-deaza-dGTP, dITP,5-methyl-dCTP, 5-hydroxy-methyl-dCTP, and further comprises one ddNTP.20. The method of claim 17 wherein, each reaction comprises at leastfour nucleoside triphosphates chosen from the group consisting of2′-F-ATP, 2′-F-CTP, 2′-F-GTP, 2′-F-UTP, 2′-F-TTP, 2′-deaza-2′-F-GTP,2′-F-ITP, and 5-methyl-2′-F-CTP, 5-hydroxymethyl-2′-F-CTP and furthercomprises one ddNTP.
 21. A method for determining the sequence of anucleic acid molecule using a mutant RNA polymerase which has a reduceddiscrimination for non-canonical versus canonical nucleotides assubstrates, comprising the steps of: a) synthesizing a nucleic acidmolecule by extending a primer, wherein at least part of the primer iscomplementary to a template molecule so as to anneal therewith, in areaction mixture containing the mutant RNA polymerase in each of fourseparate reactions, each reaction comprising at least four nucleosidetriphosphates, wherein at least one nucleoside triphosphate has anucleic acid base which is complementary to each of adenine, cytidine,guanine and uracil or thymine and a sugar with either an hydroxy or ahydrogen or a fluorine at the 2′-position, and further comprising aportion of a ddNTP, such that each of the four separate reactionscontains a ddNTP that is complementary to a different one of the fourcommon nucleic acid bases, and b) evaluating the reaction products sothat the sequence of the template molecule may be deduced.
 22. Themethod of claim 21 wherein, each reaction comprises at least fournucleoside triphosphates chosen from the group consisting of ATP, CTP,GTP, and UTP or rTTP, and further comprises one ddNTP.
 23. The method ofclaim 21 wherein, each reaction comprises at least four nucleosidetriphosphates chosen from the group consisting of dATP, dCTP, dGTP,dUTP, dTTP, 7-deaza-dGTP, dITP, 5-methyl-dCTP, 5-hydroxymethyl-dCTP andfurther comprises one ddNTP.
 24. The method of claim 21 wherein, eachreaction comprises at least four nucleoside triphosphates chosen fromthe group consisting of 2′-F-ATP, 2′-F-CTP, 2′-F-GTP, 2′-F-UTP,2′-F-TTP, 7-deaza-2′-F-GTP, 2′-F-ITP, and 5-methyl-2′-5 F-CTP,5-hydroxymethyl-2′-F-CTP and further comprises one ddNTP.
 25. The methodof claim 1, wherein a dinucleotide or trinucleotide for initiation of denovo nucleic acid synthesis is added to the reaction mixture.
 26. Themethod of claim 17, wherein at least one of the nucleoside triphosphatesin the reaction mixture is modified to contain a radioactive ornon-radioactive label.
 27. The method of claim 17, wherein the ddNTP inthe reaction mixture is modified to contain a radioactive ornon-radioactive label.
 28. The method of claim 25, wherein thedinucleotide or trinucleotide in the reaction mixture is modified tocontain a radioactive or non-radioactive label.
 29. A kit for performinga dideoxy sequencing reaction, comprising a mutant nucleic acidpolymerase which has reduced discrimination between canonical andnon-canonical nucleoside triphosphates and data or informationdescribing conditions under which the method may be performed.
 30. Amethod for determining the sequence of a nucleic acid molecule using amutant RNA polymerase which has a reduced discrimination fornon-canonical versus canonical nucleotides as substrates, comprising thesteps of: a) synthesizing a nucleic acid molecule de novo from an RNAPpromoter sequence in a reaction mixture containing a mutant RNApolymerase in each of four separate reactions, each reaction comprisingat least four nucleoside triphosphates, wherein at least one nucleosidetriphosphate has a nucleic acid base which is complementary to each ofadenine, cytidine, guanine and uracil or thymine and a sugar with ahydrogen or a fluorine at the 2′-position, and a portion of a rNTP, suchthat each of the four separate reactions contains a rNTP that iscomplementary to a different one of the four common nucleic acid bases,and b) treating the nucleic acid products of the reactions so as tobring about hydrolysis of the phosphodiester backbone at all sites wherea ribonucleotide has been incorporated, and c) evaluating the reactionproducts so that the sequence of the template molecule may be deduced.31. The method of claim 30 wherein the nucleic acid synthesis is part ofor coupled to a method for nucleic acid amplification.
 32. A kit forperforming the method of claim 30 comprising a mutant nucleic acidpolymerase which has reduced discrimination between canonical andnon-canonical nucleoside triphosphates and data or instructionsdescribing conditions under which the method of claim 30 may beperformed.
 33. A kit for performing the method of claim 31 comprising amutant nucleic acid polymerase which has reduced discrimination betweencanonical and non-canonical nucleoside triphosphates and data orinstructions describing conditions under which the method of claim 31may be performed.
 34. A method for determining the sequence of a nucleicacid molecule using a mutant RNA polymerase which has a reduceddiscrimination for non-canonical versus canonical nucleotides assubstrates, comprising the steps of: a) synthesizing a nucleic acidmolecule by extending a primer, at least part of which is sufficientlycomplementary to a template molecule so as to anneal therewith, in areaction mixture containing a mutant RNA polymerase in each of fourseparate reactions, each reaction comprising at least four nucleosidetriphosphates, wherein at least one nucleoside triphosphate has anucleic acid base which is complementary to each of adenine, cytidine,guanine and uracil or thymine and a sugar with either a hydrogen or afluorine at the 2′-position, and a portion of a rNTP, such that each ofthe four separate reactions contains a rNTP that is complementary to adifferent one of the four common nucleic acid bases in a nucleic acidmolecule, b) treating the nucleic acid products of the reactions so asto bring about hydrolysis of the phosphodiester backbone at all siteswhere a ribonucleotide has been incorporated, and c) evaluating thereaction′products using any of the methods common in the art forseparating and detecting reaction products of sequencing reactions sothat the sequence of the template molecule may be deduced.
 35. Themethod of claim 34 wherein the nucleic acid synthesis is part of orcoupled to a method for nucleic acid amplification.
 36. A kit forperforming the method of claim 34 comprising a mutant nucleic acidpolymerase which has reduced discrimination between canonical andnon-canonical nucleoside triphosphates and data or instructionsdescribing conditions under which the method of claim 34 may beperformed.
 37. A kit for performing the method of claim 35 comprising amutant nucleic acid polymerase which has reduced discrimination betweencanonical and non-canonical nucleoside triphosphates and data orinstructions describing conditions under which the method of claim 35may be performed.
 38. The method of claim 1 wherein the nucleic acidsynthesis is part of or coupled to a method for nucleic acidamplifications.
 39. A kit for performing the method of claim 38comprising a mutant nucleic acid polymerase which has reduceddiscrimination between canonical and non-canonical nucleosidetriphosphates and data or instructions describing conditions under whichthe method of claim 38 may be performed.
 40. A kit for performing apartial ribo-substitution reaction comprising a mutant nucleic acidpolymerase which has reduced discrimination between canonical andnon-canonical nucleoside triphosphates and data or informationdescribing conditions under which the method may be performed.